N-substituted indenoisoquinolines and syntheses thereof

ABSTRACT

N-Substituted indenoisoquinoline compounds, and pharmaceutical formulations of N-substituted indenoisoquinoline compounds are described. Also described are processes for preparing N-substituted indenoisoquinoline compounds. Also described are methods for treating cancer in mammals using the described N-substituted indenoisoquinoline compounds or pharmaceutical formulations thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 (e) of U.S.Provisional Application Ser. No. 60/736,471, filed Nov. 14, 2005, andU.S. Provisional Application Ser. No. 60/808,699, filed May 26, 2006,the disclosures of which are hereby incorporated herein by reference.

TECHNICAL FIELD

The invention described herein pertains to N-substitutedindenoisoquinoline compounds. The invention described herein alsopertains to methods for treating cancer in mammals usingindenoisoquinoline compounds.

BACKGROUND

The control and cure of cancer represents one of our most challenginghealth problems. The treatment of cancer can be approached by severalmodes of therapy including surgery, radiation, chemotherapy or acombination of any of these treatments. Chemotherapy continues to be anindispensable therapy for inoperable or metastatic forms of the disease.Thus, the discovery of compounds specifically targeting cancer cells, orthe cellular mechanisms involved in the proliferation of cancer cells,can provide significant advancement in the eradication or control ofcancer.

The selection of compounds having effective anticancer activity iscomplicated by the still limited knowledge of cancer cell biology andbiochemistry. Therefore, development of new effective anti-cancer agentsremains heavily dependent on screening of new compounds for cytotoxicactivity. Antineoplastic drug candidates exhibit enhanced cytotoxicityagainst cancer cells relative to normal cells. Methods of screening foranticancer activity have focused on several targets, (1) the ability ofa compound to inhibit tumor growth and/or progression in animal studies;(2) inhibition of cell growth/proliferation in cell lines of cancerousorigin; and (3) inhibition of intracellular processes necessary for thegrowth or propagation of cancer cells.

The mouse L1210 leukemia cell line was initially the preferred modelsystem used for screening compounds for anti-cancer activity. However,the P388 murine leukemia system was found to be more sensitive andpredictive than L1210 leukemia system; it has been used as a primaryscreen during the past decade. Systematic screening for compoundsexhibiting toxicity to these two cell lines has resulted in theisolation of a large number of active natural products. However, theanticancer activities of these compounds were predominantly forleukemia, lymphoma and a few rare tumors. Low clinical efficacy, or thelack of clinical efficacy of known chemotherapeutics against slowergrowing solid tumors, is a serious concern.

Considering the diversity of cancer in terms of cell type, morphology,growth rate and other cellular characteristics, the U.S. National CancerInstitute (NCI) has developed a disease-oriented approach to anticanceractivity screening (M. R. Boyd, in “Principle of Practice of Oncology”J. T. Devita, S. Hellman, S. A. Rosenberg (Eds.) Vol. 3, PPO Update, No.10, 1989). This in vitro prescreening system ds based on the measurementof anticancer cytotoxicity against human cancer cell line panelsconsisting of approximately 60 cell lines of major human cancers(including leukemia, and slower growing tumor cells such as lung, colon,breast, skin, kidney, etc.) and is referred hereinafter as “COMPARE”screening. An important advantage of the new in vitro screening panelsis the opportunity to facilitate identification of compounds that areselectively more cytotoxic to cells of certain types of cancers, thusincreasing the ability to select compounds for further study withrespect to specific diseases.

Anticancer agents are known to act through a variety of mechanisms todestroy or inhibit the proliferation of cancer cells. For example, someagents are antimetabolites which act as false substrates in thebiochemical processes of cancer cells. One compound which has thismechanism of action is methotrexate, an analog of folic acid, whichfunctions in part by binding to dihydrofolate reductase, therebypreventing the formation of guanine and adenine from the folic acidprecursor molecule. Thus, methotrexate inhibits the ability of cancercells to construct DNA by inhibiting the proper metabolism of folicacid.

Other anticancer agents act by alkylating DNA strands, thereby producingdefects in the normal double helical structure of the DNA molecule. Thisalkylation may cause the formation of breaks and inappropriate linksbetween (or within) strands of DNA. Such disruption of the DNAstructure, if not repaired by intracellular repair mechanisms, impairsthe cell's ability to replicate its DNA. Examples of alkylatinganticancer agents are cyclophosphamide and chlorambucil.

Some anticancer agents target the intracellular mechanisms involved inreplication of the DNA strand itself. Replication of a cell's geneticmaterial requires a means to pull the DNA double helix apart into twostrands. This separation is typically accomplished by the enzymetopoisomerase I. Disruption of the function of this enzyme results inDNA strand breaks in cells that are dividing, thereby causing the deathof the dividing cell. Because cancer cells grow and reproduce at a muchfaster rate than normal cells, they are more vulnerable to topoisomeraseI inhibition than are normal cells. Thus, agents that inhibittopoisomerase I are known to be potent anticancer agents. The drugcamptothecin was shown to be an inhibitor of topoisomerase I and apotent anticancer agent. However, it has been observed that camptothecinmay produce toxic side effects. In addition, the effectiveness ofcamptothecin is hampered by both the instability of the molecule itself,resulting in lactone ring opening, and the reversible nature of theinhibition, allowing impacted cells to recover. Therefore, the searchfor potent inhibitors of topoisomerase I continues.

SUMMARY OF THE INVENTION

Described herein are N-substituted indenoisoquinoline compounds, andmore specifically, substituted 11H-indeno[1,2-c]isoquinoline compounds,including dimers of such substituted 11H-indeno[1,2-c]isoquinolinecompounds formed with a divalent linker. The compounds described hereinmay be useful for treating cancer. Also described herein arepharmaceutical compositions of such compounds, processes for preparingN-substituted indenoisoquinoline compounds, and methods for treatingcancer by administering therapeutically effective amounts of suchsubstituted indenoisoquinoline compounds alone or as pharmaceuticalcompositions.

In one illustrative embodiment, novel compounds of formula I aredescribed

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein:

m is an integer from 0 to about 6;

R⁶ is selected from haloalkyl, halocycloalkyl, hydroxy, alkoxy,cycloalkoxy, haloalkoxy, halocycloalkoxy, optionally substitutedheteroaryl, aryloxy, heteroaryloxy, and heteroarylamino, acyloxy,haloacyloxy, amino, alkyl and dialkylamino, trialkylammonium,hydroxyalkylamino, bis(hydroxyalkyl)amino, hydroxyalkylaminoallylamino,heteroarylalkylaminoalkylamino, acylamino, hydroxylamino, alkoxylamino,acyloxylamino, cycloalkyl, heterocyclyl, heterocyclylamino, alkynyl,acyl, urethanyl, cyano, nitro, azido, thio, alkylsulfonyl, sulfonic acidand derivatives thereof, carboxylic acid and derivatives thereof, andphosphonic acid and derivatives thereof; and

R^(a) and R^(d) each independently represent hydrogen, or one or moreoptional and independently selected monovalent and divalentsubstituents.

In one aspect, R^(a) represents 1-4 substituents each of which isindependently selected from the group consisting of halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, cyano,nitro, optionally substituted alkylthio, optionally substitutedalkylsulfonyl, carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof; or R^(a) represents 2-4 substituents where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle,and where the remaining substituents, in cases where R^(a) represents3-4 substituents, are each independently selected from the groupconsisting of halo, hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy, cyano, nitro, optionally substituted alkylthio,optionally substituted alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof.

In another aspect, R^(d) represents 1-4 substituents each of which isindependently selected from the group consisting of halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, cyano,nitro, optionally substituted alkylthio, optionally substitutedalkylsulfonyl, carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof; or R^(d) represents 2-4 substituents where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle,and where the remaining substituents, in cases where R^(a) represents3-4 substituents, are each independently selected from the groupconsisting of halo, hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy, cyano, nitro, optionally substituted alkylthio,optionally substituted alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof.

In another illustrative embodiment, m is the integer 0. In anotherembodiment, m is an integer from 1 to about 6, and R⁶ is selected fromhalo, haloalkyl, halocycloalkyl, alkoxy, cycloalkoxy, haloalkoxy,halocycloalkoxy, optionally substituted heteroaryl, aryloxy,heteroaryloxy, and heteroarylamino, acyloxy, haloacyloxy, amino,dialkylamino, trialkylammonium, bis(hydroxyalkyl)amino,hydroxyalkylaminoalkylamino, heteroarylalkylaminoalkylamino, acylamino,hydroxylamino, alkoxylamino, acyloxylamino, cycloalkyl, heterocyclyl,heterocyclylamino, alkynyl, acyl, urethanyl, cyano, nitro, azido, thio,alkylsulfonyl, sulfonic acid and derivatives thereof, carboxylic acidand derivatives thereof, and phosphonic acid and derivatives thereof.

In another illustrative embodiment, R⁶ includes a water soluble orhydrophilic functional group. In one aspect, R⁶ includes an optionallysubstituted aminoalkyl. In another aspect, R⁶ includes an alkyl groupsubstituted with optionally substituted heteroaryl, heteroaryloxy, orheteroarylamino, amino, dialkylamino, trialkylammonium,bis(hydroxyalkyl)amino, hydroxyalkylaminoalkylamino,heteroarylalkylaminoalkylamino, acylamino, hydroxylamino, alkoxylamino,acyloxylamino, heterocyclyl, heterocyclylamino, nitro, or azido.

In another illustrative embodiment, R^(a) represents 1-4 substituentseach of which is independently selected from the group consisting ofhalo, hydroxy, optionally substituted alkyl, optionally substitutedalkoxy, cyano, nitro, optionally substituted alkylthio, optionallysubstituted alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof.

In another illustrative embodiment, R^(a) represents 2-4 substituentswhere 2 of said substituents are adjacent substituents and are takentogether with the attached carbons to form an optionally substitutedheterocycle, and where any remaining substituents are each independentlyselected from the group consisting of halo, hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy, cyano, nitro,optionally substituted alkylthio, optionally substituted alkylsulfonyl,carboxylic acid and derivatives thereof, and sulfonic acid andderivatives thereof.

In another illustrative embodiment, R^(d) represents 1-4 substituentseach of which is independently selected from the group consisting ofhalo, hydroxy, optionally substituted alkyl, optionally substitutedalkoxy, cyano, nitro, optionally substituted alkylthio, optionallysubstituted alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof.

In another illustrative embodiment, R^(d) represents 2-4 substituentswhere 2 of said substituents are adjacent substituents and are takentogether with the attached carbons to form an optionally substitutedheterocycle, and where any remaining substituents are each independentlyselected from the group consisting of halo, hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy, cyano, nitro,optionally substituted alkylthio, optionally substituted alkylsulfonyl,carboxylic acid and derivatives thereof, and sulfonic acid andderivatives thereof.

In another illustrative embodiment, m is an integer from 0 to about 6,and R⁶ is selected from the group consisting of haloalkyl,halocycloalkyl, hydroxy, alkoxy, cycloalkoxy, haloalkoxy,halocycloalkoxy, optionally substituted heteroaryl, aryloxy,heteroaryloxy, and heteroarylamino, acyloxy, haloacyloxy, amino, alkyland dialkylamino, trialkylammonium, bis(hydroxyalkyl)amino,hydroxyalkylaminoalkylamino, heteroarylalkylaminoalkylamino, acylamino,hydroxylamino, alkoxylamino, acyloxylamino, cycloalkyl, heterocyclyl,heterocyclylamino, alkynyl, acyl, urethanyl, cyano, nitro, azido, thio,alkylsulfonyl, sulfonic acid and derivatives thereof, carboxylic acidand derivatives thereof, and phosphonic acid and derivatives thereof;provided that when R⁶ is hydroxy, alkylamino, or hydroxyalkylamino, m isthe integer 0.

In another illustrative embodiment, novel compounds of formula II aredescribed

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein:

R^(a), R^(d), R^(a′), and R^(d′) each independently represent 4substituents, all of which are independently selected from the groupconsisting of hydrogen, halo, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, cyano, nitro, optionally substitutedalkylthio, optionally substituted alkylsulfonyl, carboxylic acid andderivatives thereof, and sulfonic acid and derivatives thereof; or where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle;and

X is a divalent linker comprising one or more divalent radicals selectedfrom —(CR¹R²), —(NR¹)— and —O—, where R¹ and R² are independentlyselected in each occurrence from hydrogen, alkyl, and acyl, providingthat the divalent linker does not include —O—O—. In one aspect, ifpresent, each divalent —(NR¹)— and —O— is separated by at least onedivalent radical (—CR¹R²)—. In another aspect, each R¹ and R² ishydrogen.

In another illustrative embodiment, X is a group having the generalstructure —(CH₂)_(n)—[(CH₂)_(x)—NR¹—(CH₂)_(y)]_(z)—(NR²)_(p)—(CH₂)_(q)—,where n is 0 or 1, x and y are integers independently ranging from 1 toabout 4, z is an integer ranging from 1 to about 4, p is 0 or 1, q is 0or an integer ranging from 1 to about 2, and where R¹ and R² areindependently selected in each instance from hydrogen, methyl,t-butyloxycarbonyl, benzyloxycarbonyl, and fluorenylmethoxycabonyl, orR¹ and R² and any adjacent R² together with the attached nitrogens forma heterocycle.

In another illustrative embodiment, indenoisoquinoline compounds offormulae I and II described herein are useful for treating cancer ortumors. In one aspect, compounds described herein exhibit the activityof stabilizing DNA-topoisomerase I cleavage complexes throughintercalation at the DNA cleavage site, resulting in inhibition of thereligation reaction. See, for example, Kohlhagen, G.; Paull, K.;Cushman, M.; Nagafuji, P.; Pommier, Y. Protein-Linked DNA Strand BreaksInduced by NSC 314622, a Novel Noncamptothecin Topoisomerase I PoisonMol. Pharmacol. 1998, 54, 50-58; Pommier, Y.; Pourquier, P.; Fan, Y.;Strumberg, D. Mechanism of Action of Eukaryotic DNA Topoisomerases andDrugs Targeted to the Enzyme Biochem. Biophys. Acta. 1998, 1400, 83-105;Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin Jr., A.B.; Stewart, L. The Mechanism of Topoisomerase I Poisoning by aCamptothecin Analog Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15387-15392,the disclosures of each of which are incorporated herein by reference.As inhibitors of the DNA religation reaction occurring after DNAcleavage by topoisomerase 1, compounds described herein may beclassified as “top 1 poisons,” and may exhibit biological andpharmacological activity similar to that observed with camptothecins. Inanother aspect, indenoisoquinoline compounds of formulae I and IIdescribed herein may be efficacious against various types of humancancers. In another aspect, indenoisoquinoline compounds of formulae Iand II described herein may be chemically more stable than camptothecin.In yet another aspect, indenoisoquinoline compounds of formulae I and IIdescribed herein may have unique DNA binding site selectivities relativeto camptothecin.

In another illustrative embodiment, methods for treating human cancersare described. In one aspect of the methods described herein, thecancers are attributable to abnormally fast cell growth, reproduction,and/or proliferation. In another aspect, the cancers treatable bycompounds of formulae I and II are responsive to enzyme inhibition, suchas inhibition of topoisomerase 1.

In another illustrative embodiment, processes for preparingindenoisoquinoline compounds of formula I and II are described. In oneembodiment, the processes include preparing an intermediatebenz[d]indeno[1,2-b]pyran-5,11-dione of the formula III

where the process comprises reacting a 2-carboxybenzaldehyde compoundand a phthalide compound of respective formulae

followed by acidic treatment in benzene under reflux or treatment with asuitable coupling reagent such as dicyclohexylcarbodiimide withdimethyaminopyridine, wherein R^(a) and R^(d) are as defined herein forcompounds of formulae I and II.

In another illustrative embodiment, processes are described herein forpreparing compounds of formulae I and II comprising the steps of (i)reacting an R^(a)-substituted hydroxy phthalide compound and anR^(d)-substituted phthalide compound of respective formulae

in the presence of a base, then (ii) treating the mixture with an acidto prepare the intermediate benz[d]indeno[1,2-b]pyran-5,11-dionedescribed above, wherein R^(d) and R^(d) are as defined herein forcompounds of formulae I and II.

In another illustrative embodiment, processes are described herein forpreparing compounds of formulae I and II comprising the steps ofreacting a benz[d] indeno[1,2-b]pyran-5,11-dione of the formula

with a primary amine of the formula R⁶—(CH₂)_(m)—NH₂, wherein R^(a),R^(d), m, and R⁶ are as defined herein for compounds of formulae I andII. In one embodiment, the primary amine is illustratively4-(2-aminoethyl)morpholine, 1-(3-aminopropyl)imidazole,N-(3-aminopropyl)-N,N-dimethylamine, 4-(hydroxy)butylamine,3-(bromo)propylamine, a mono-Boc-protected diamine, and the like. It isappreciated that although chloroform at room temperature will suffice asthe solvent for most primary amines, when a primary amine such as amono-Boc-protected diamine, for example, is used to form the lactam froma benz[d]indeno[1,2-b]pyran-5,11-dione, chloroform at reflux may be usedas the solvent. It is further appreciated that an indenoisoquinolinecompound for which the integer m is not 0, and wherein R⁶ is halo,azido, or cyano, for example, may be further elaborated throughdisplacement of the halo, azido, or cyano functionality, respectively,with a variety of nucleophiles.

In another illustrative embodiment, processes are described herein forpreparing compounds of formulae I and II comprising the steps ofreacting an optionally substituted homophthalic anhydride and anoptionally substituted Schiff base of respective formulae

followed by subjecting the resulting carboxylic acid to oxidativeFriedel-Crafts ring closure with thionyl chloride and aluminum chloride,wherein R^(a), R^(d), m, and R⁶ are as defined herein for compounds offormulae I and II.

In another illustrative embodiment, processes are described herein forpreparing compounds of formulae I and II comprising the steps ofreacting a benz[d]indeno[1,2-b]pyran-5,11-dione of the formula

with a polyamine of the formulaNH₂—(CH₂)_(n)—[(CH₂)_(x)—NR¹—(CH₂)_(y)]_(z)—(NR²)_(p)—(CH₂)_(q)—NH₂,where R¹, R², n, x, y, z, p, q, R^(a), and R^(d) are as defined hereinfor compounds of formula II. In one embodiment, the polyamine isillustratively N,N-bis(2-aminoethyl)amine, N,N-bis(3-aminopropyl)amine,N-(2-aminoethyl)-N-(3-aminopropyl)-amine,N,N′-bis(2-aminoethyl)-1,3-propanediamine,N,N′-bis(3-aminopropyl)-1,3-propanediamine and the like.

It is to be understood that each of the aspects of the variousillustrative embodiments described herein may be modified and/orcombined as additional illustrative embodiments. For example,illustrative embodiments of the compounds of formula II may includethose aspects wherein an unsubstituted, symmetricalbisindenoisoquinoline is present, as reflected in the use of abenz[d]indeno[1,2-b]pyran-5,11-dione where R^(a) and R^(d) are eachhydrogen. Further, illustrative embodiments of the compounds of formulaII may include those aspects wherein a substituted, symmetricalbisindenoisoquinoline is present, as reflected in the use of abenz[d]indeno[1,2-b]pyran-5,11-dione where, for example, R^(a) is2,3-dimethoxy and R^(d) is hydrogen, or where, for example, R^(a) is3-nitro and R^(d) is hydrogen. In addition, illustrative embodiments ofthe compounds of formula II may include those aspects wherein asubstituted, unsymmetrical bisindenoisoquinoline is present, asreflected in the use of a mixture of two differentially substitutedbenz[d]indeno[1,2-b]pyran-5,11-diones to prepare a dimer such as

wherein Ra, Rd, and X are as defined herein for compounds of formula II,and wherein Ra≠H and/or Rd≠H.

DETAILED DESCRIPTION

In one illustrative embodiment, novel compounds of formula I aredescribed

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein:

m is an integer from 0 to about 6; and R⁶ is selected from haloalkyl,halocycloalkyl, hydroxy, alkoxy, cycloalkoxy, haloalkoxy,halocycloalkoxy, optionally substituted heteroaryl, aryloxy,heteroaryloxy, and heteroarylamino, acyloxy, haloacyloxy, amino, alkyland dialkylamino, trialkylammoniurn, hydroxyalkylamino,bis(hydroxyalkyl)amino, hydroxyalkylaminoalkylamino,heteroarylalkylaminoalkylamino, acylamino, hydroxylamino, alkoxylamino,acyloxylamino, cycloalkyl, heterocyclyl, heterocyclylamino, alkynyl,acyl, urethanyl, cyano, nitro, azido, thio, alkylsulfonyl, sulfonic acidand derivatives thereof, carboxylic acid and derivatives thereof, andphosphonic acid and derivatives thereof;

R^(a) represents 1-4 substituents each of which is independentlyselected from the group consisting of hydrogen, halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, cyano,nitro, optionally substituted alkylthio, optionally substitutedalkylsulfonyl, carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof; or R^(a) represents 2-4 substituents where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle,and where the remaining substituents, in cases where R^(a) represents3-4 substituents, are each independently selected from the groupconsisting of hydrogen, halo, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, cyano, nitro, optionally substitutedalkylthio, optionally substituted alkylsulfonyl, carboxylic acid andderivatives thereof, and sulfonic acid and derivatives thereof; and

R^(d) represents 1-4 substituents each of which is independentlyselected from the group consisting of hydrogen, halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, cyano,nitro, optionally substituted alkylthio, optionally substitutedalkylsulfonyl, carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof; or R^(d) represents 2-4 substituents where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle,and where the remaining substituents, in cases where represents 3-4substituents, are each independently selected from the group consistingof hydrogen, halo, hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy, cyano, nitro, optionally substituted alkylthio,optionally substituted alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof.

As used herein, the term “alkyl” refers to a saturated monovalent chainof carbon atoms, which may be optionally branched. It is understood thatin embodiments that include alkyl, illustrative variations of thoseembodiments include lower alkyl, such as C₁-C₆, C₁-C₄ alkyl, methyl,ethyl, propyl, 3-methylpentyl, and the like.

As used herein, the term “cycloalkyl” refers to a monovalent chain ofcarbon atoms, a portion of which forms a ring. It is understood that inembodiments that include cycloalkyl, illustrative variations of thoseembodiments include lower cycloalkyl, such as C₃-C₈, C₃-C₆ cycloalkyl,cyclopropyl, cyclohexyl, 3-ethylcyclopentyl, and the like.

As used herein, the term “alkenyl” refers to an unsaturated monovalentchain of carbon atoms including at least one double bond, witch may beoptionally branched. It is understood that in embodiments that includealkenyl, illustrative variations of those embodiments include loweralkenyl, such as C₂-C₆, C₂-C₄ alkenyl.

As used herein, the term “cycloalkenyl” refers to an unsaturatedmonovalent chain of carbon atoms, a portion of which forms a ring. It isunderstood that in embodiments that include cycloalkenyl, illustrativevariations of those embodiments include lower cycloalkenyl, such asC₃-C₈, C₃-C₆ cycloalkenyl.

As used herein, the term “alkylene” refers to a saturated bivalent chainof carbon atoms, which may be optionally branched. It is understood thatin embodiments that include alkylene, illustrative variations of thoseembodiments include lower alkylene, such as C₂-C₄, alkylene, methylene,ethylene, propylene, 3-methylpentylene, and the like.

As used herein, the term “heterocycle” refers to a monovalent chain ofcarbon and heteroatoms, wherein the heteroatoms are selected fromnitrogen, oxygen, and sulfur, a portion of which, including at least oneheteroatom, form a ring, such as aziridine, pyrrolidine, oxazolidine,3-methoxypyrrolidine, 3-methylpiperazine, and the like.

It is to be understood that each of alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkylene, and heterocyclyl may be optionally substitutedwith independently selected groups such as alkyl, haloalkyl,hydroxyalkyl, aminoalkyl, carboxylic acid and derivatives thereof,including esters, amides, and nitriles, hydroxy, alkoxy, acyloxy, amino,alkyl and dialkylamino, acylamino, thio, and the like, and combinationsthereof.

As used herein, the term “optionally substituted aryl” refers to anaromatic mono or polycyclic ring of carbon atoms, such as phenyl,naphthyl, and the like, which may be optionally substituted with one ormore independently selected substituents, such as halo, hydroxy, amino,alkyl or dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro, and thelike.

As used herein, the term “optionally substituted heteroaryl” refers toan aromatic mono or polycyclic ring of carbon atoms and at least oneheteroatom selected from nitrogen, oxygen, and sulfur, such aspyridinyl, pyrimidinyl, indolyl, benzoxazolyl, and the like, which maybe optionally substituted with one or more independently selectedsubstituents, such as halo, hydroxy, amino, alkyl or dialkylamino,alkoxy, alkylsulfonyl, cyano, nitro, and the like.

As used herein, the term “acyl” refers to hydrogen, alkyl, cycloalkyl,alkenyl, cycloalkenyl, heterocyclyl, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl, andoptionally substituted heteroarylalkyl attached as a substituent througha carbonyl (C═O) group, such as formyl, acetyl, pivalolyl, benzoyl,phenacetyl, and the like.

As used herein, the terms “dialkylamino,” “dialkylammonium,” and“trialkyammonium” refer to amino substituted with alkyl groups, whereeach alkyl group is independently selected, and illustratively includesdimethylamino, methylethylamino, diisopropylethylammonium,benzyldimethylammonium, benzyldiisopropylamrnoniutn, and the like.

As used herein, the terms “protected hydroxy” and “protected amino”refer to hydroxy and amino groups, respectively, that are protected witha protecting group. It is to be understood that such protecting groupsare conventional and routinely selected to allow a synthetic or chemicaltransformation to be performed in a manner that the hydroxy group oramino group does not interfere with or is not changed by the syntheticor chemical transformation performed. Illustrative, but not exclusive,examples of such protecting groups may be found in Greene & Wuts“Protective Groups in Organic Synthesis,” 2d Ed., John Wiley & Sons, NewYork, 1991, the disclosure of which is incorporated herein by reference.Further illustrative of such protecting groups are those particularlysuited for protecting phenols and catechols, and analogs and derivativesthereof.

In one illustrative aspect of the compounds of formula I, R⁶ isdialkylamino, including dimethylamino, azido, poly(hydroxyalkyl)amino,hydroxyalkylaminoalkylamino, polyhydroxyalkylaminoalkylamino,hydroxyalkyl(alkylamino), heteroaryl, or a combination thereof. Inanother aspect, R⁶ is selected from the formulae

each of which may be optionally substituted. In another aspect, m is 2,3, or 4.

In another aspect of the compounds of formula I, R⁶ is alkyl substitutedwith amino, dialkylamino, trialkylammonium, poly(hydroxyalkyl)amino,hydroxyalkylaminoalkylamino, (polyhydroxy)alkylaminoalkylamino,heteroaryl, azido, hydroxyalkyl(alkylamino), and combinations thereof.In another aspect, R⁶ is substituted C₁-C₄ alkyl. In another aspect, R⁶is substituted C₃ alkyl.

In another illustrative embodiment of the compounds of formula I, R^(a)represents one or more substituents selected from optionally substitutedalkoxy. In one aspect, R^(a) represents at least two adjacentsubstituents taken together to form alkylenedioxy. In anotherembodiment, R^(a) represents one or more substituents selected fromhalo, hydroxy, amino, alkyl and dialkylamino, nitroso, nitro,hydroxylamino, alkoxylamino, and cyano. In another embodiment of thecompounds of formula I, R^(d) represents one or more substituentsselected from optionally substituted alkoxy. In one aspect, R^(d)represents at least two adjacent substituents taken together to formalkylenedioxy. In another embodiment, R^(d) represents one or moresubstituents selected from halo, amino, alkyl and dialkylamino, nitroso,nitro, and cyano.

In another illustrative embodiment of the compounds of formula I, R⁶ isalkyl substituted with amino, dialkylamino, trialkylammonium,poly(hydroxyalkyl)amino, hydroxyalkylaminoalkylamino,(polyhydroxy)alkylaminoalkylamino, heteroaryl, azido,hydroxyalkyl(alkylamino), and combinations thereof. In another aspect,R⁶ is substituted C₁-C₄ alkyl. In another aspect, R⁶ is substituted C₃alkyl. In another aspect, R^(a) represents one or more substituentsselected from optionally substituted alkoxy. In another aspect, R^(a)represents at least two adjacent substituents taken together to formalkylenedioxy. In another aspect, R^(a) represents one or moresubstituents selected from halo, hydroxy, amino, alkyl and dialkylamino,nitroso, nitro, hydroxylamino, alkoxylamino, and cyano. In anotheraspect, R^(d) represents one or more substituents selected fromoptionally substituted alkoxy. In another aspect, R^(d) represents atleast two adjacent substituents taken together to form alkylenedioxy. Inanother aspect, R^(d) represents one or more substituents selected fromhalo, amino, alkyl and dialkylamino, nitroso, nitro, and cyano.

In another illustrative embodiment, indenobenzopyran compounds offormula III are described, where R^(a) and R^(d) are as defined in thecompounds of formulae I and II. These compounds may be used to preparecompounds of formulae I and II according to the processes describedherein. In one embodiment, compounds 4a-4s are described, as shown inthe following table. Compounds 4a-4s were prepared by the processesdescribed herein comprising the steps of preparing and cyclizing theadduct of an optionally substituted 2-carboxybenzaldehyde and anoptionally substituted phthalide as described herein.

(III)

Compound R^(a) R^(d) 4a 2,3-(MeO)₂ H 4b 2,3-(OCH₂O) H 4c 3-NO₂ H 4d H H4e 2,3-(MeO)₂ 8,9-(OCH₂O) 4f 2,3-(MeO)₂ 8,9-(MeO)₂ 4g 2,3-(MeO)₂7,8,9-(MeO)₃ 4h 2,3-(OCH₂O) 8,9-(MeO)₂ 4i 2,3-(OCH₂O) 8,9-(OCH₂O) 4j2,3-(OCH₂O) 7,8,9-(MeO)₃ 4k 1,2,3-(MeO)₃ 8,9-(MeO)₂ 4l 1,2,3-(MeO)₃8,9-(OCH₂O) 4m 1,2,3-(MeO)₃ 7,8,9-(MeO)₃ 4n 1,4-(MeO)₂ 8,9-(MeO)₂ 4o1,4-(MeO)₂ 8,9-(OCH₂O) 4p 1,4-(MeO)₂ 7,8,9-(MeO)₃ 4q 2,3,4-(MeO)₃8,9-(MeO)₂ 4r 2,3,4-(MeO)₃ 8,9-(OCH₂O) 4s 2,3,4-(MeO)₃ 7,8,9-(MeO)₃

In another illustrative embodiment, novel indenoisoquinoline compounds5a-5z, 5aa-5az, and 5ba-5bs are described. These compounds were preparedby the processes described herein comprising the steps of preparing thecorresponding benz[d]indeno[1,2-b]pyran-5,11-dione and converting thelactone into the corresponding lactam with a suitable primary amine, orby condensing an optionally substituted homophthalic anhydride and anoptionally substituted Schiff base, as described herein.

(I)

Com- pound R^(a) R^(d) m R⁶ 5a 2,3-(MeO)₂ H  3 (Me)₂N 5b 2,3-(OCH₂O) H 3 (Me)₂N 5c 3-NO₂ H  3 (Me)₂N 5d H H  3 (Me)₂N 5e 2,3-(MeO)₂ H  3imidazol-1-yl 5f 3-NO₂ H  3 imidazol-1-yl 5g H H  3 imidazol-1-yl 5h2,3-(MeO)₂ H  2 morpholin-1-yl 5i 3-NO₂ H  2 morpholin-1-yl 5j H H  2morpholin-1-yl 5k 2,3-(MeO)₂ 8,9-(OCH₂O)  3 CF₃CO₂ 5l 2,3-(MeO)₂8,9-(OCH₂O)  3 imidazol-1-yl•2HCl 5m 2,3-(MeO)₂ 8,9-(OCH₂O)  3pyrazol-1-yl 5n 2,3-(MeO)₂ 8,9-(OCH₂O)  3 triazol-1-yl•HCl 5o 2,3-(MeO)₂8,9-(OCH₂O)  3 thiazol-2-ylamino•2HCl 5p 2,3-(MeO)₂ 8,9-(OCH₂O)  3piperazin-1-yl•2HCl 5q 2,3-(MeO)₂ 8,9-(OCH₂O)  3 morpholin-1-yl 5r2,3-(MeO)₂ 8,9-(OCH₂O)  3 thiomorpholin-1-yl 5s 2,3-(MeO)₂ 8,9-(OCH₂O) 3 3-hydroxypiperidin-1-yl•HCl 5t 2,3-(MeO)₂ 8,9-(OCH₂O)  31-methylpiperazin-1-yl•2HCl 5u 2,3-(MeO)₂ 8,9-(OCH₂O)  34-aminopiperidin-1-yl•2HCl 5v 2,3-(MeO)₂ 8,9-(OCH₂O)  3homopiperazin-1-yl•2HCl 5w 2,3-(MeO)₂ 8,9-(OCH₂O)  34-(hydroxyethyl)piperazin-1-yl 5x 2,3-(MeO)₂ 8,9-(OCH₂O)  3morpholinylethylamino 5y 2,3-(MeO)₂ 8,9-(OCH₂O)  3 bromo 5z H H  0 —NH₂5aa H H  2 —NHBoc 5ab H H  3 —NHBoc 5ac H H  4 —NHBoc 5ad H H  5 —NH₂5ae H H  6 —NH₂ 5af H H  7 —NHBoc 5ag H H  8 —NHBoc 5ah H H  9 —NHBoc5ai H H 10 —NHBoc 5aj H H 11 —NHBoc 5ak H H 12 —NHBoc 5al H H  2 —NH₃⁺Cl⁻ 5am H H  3 —NH₃ ⁺Cl⁻ 5an H H  4 —NH₃ ⁺Cl⁻ 5ao H H  5 —NH₃ ⁺Cl⁻ 5apH H  6 —NH₃ ⁺Cl⁻ 5aq H H  7 —NH₃ ⁺Cl⁻ 5ar H H  8 —NH₃ ⁺Cl⁻ 5as H H  9—NH₃ ⁺Cl⁻ 5at H H 10 —NH₃ ⁺Cl⁻ 5au H H 11 —NH₃ ⁺Cl⁻ 5av H H 12 —NH₃ ⁺Cl⁻5aw H H  1 2-pyridyl 5ax H H  1 3-pyridyl 5ay H H  2 2-pyridyl 5az H H 2 3-pyridyl 5ba 3-NO₂ 9-MeO  3 chloro 5bb 3-NO₂ H  3 bromo 5bc H 9-MeO 3 chloro 5bd 3-NO₂ 9-MeO  3 iodo 5be H 9-MeO  3 iodo 5bf H 9-MeO  3azido 5bg H 9-MeO  3 —NH₃ ⁺Cl⁻ 5bh H H  3 azido 5bi 3-NO₂ H  3morpholin-1-yl 5bj 3-NO₂ 9-MeO  3 morpholin-1-yl 5bk H 9-MeO  3morpholin-1-yl 5bl 3-NO₂ H  3 —NH—CH₂—CH₂—OH•HCl 5bm 3-NO₂ 9-MeO  3—NH—CH₂—CH₂—OH•HCl 5bn H 9-MeO  3 —NH—CH₂—CH₂—OH•HCl 5bo H H  3—NH—CH₂—CH₂—OH•HCl 5bp 3-NO₂ 9-MeO  3 (Me)₂N 5bq H 9-MeO  3 (Me)₂N 5br3-NO₂ 9-MeO  3 imidazol-1-yl 5bs H 9-MeO  3 imidazol-1-yl

It is appreciated that compounds 5a-5z, 5aa-5az, and 5ba-5bs may bechemically more stable than camptothecin, owing, at least in part, tothe absence of a lactone ring, such as is found in camptothecin. See,(a) Jaxel, C.; Kohn, K. W.; Wani, M. C.; Pommier. Y. Structure-ActivityStudy of the Actions of Camptothecin Derivatives on MammalianTopoisomerase 1: Evidence for a Specific Receptor Site and a Relation toAntitumor Activity Cancer. Rev. 1989, 49, 1465-1469. (b) Minanri, H.;Beijnen, J. H.; Verweij, J.; Ratain, M. J. Limited Sampling Model forthe Area under the Concentration Time Curve of Total Topotecan Clin.Cancer Res. 1996, 2, 43.46. (c) Danks, M. K.; Pawlik, C. A.; Whipple, D.O.; Wolverton, J. S. Intermittant Exposure of Medulloblastoma Cells toTopotecan Produces Growth Inhibition equivalent to Continuous ExposureCurr. Topics Med. Chem. 1997, 3, 1731-1738. (d) Haas. N. B.; LaCreta, F.P.; Walczak, J.; Hudes, G. R.; Brennan, J. M.; Ozols, R. F.; O'Dwyer, P.J. Phasel/Pharmaco-kinetic Study of Topotecan by 24-Hour ContinuousInfusion Weekly Cancer Res. 1994, 54, 1220-1226, the disclosures ofwhich are incorporated herein by reference. It is further appreciatedthat compounds 5a-5z, 5aa-5az, and 5ba-5bs may be efficacious againstvarious types of human cancers. It is also appreciated that compounds5a-5z, 5aa-5az, and 5ba-5bs may have unique DNA binding siteselectivities relative to camptothecin.

In another illustrative embodiment, processes for preparingunsubstituted benz[d]indeno[1,2-b]pyran-5,11-dione 4d are described. Inone aspect, indenobenzopyran 4d may be prepared as shown in Scheme 1,wherein condensation of 2-carboxybenzaldehyde Id and phthalide 2d inmethanol/ethyl acetate with sodium methoxide (step (a)) generates anintermediate 3d, which can be isolated and subsequently cyclized inacidified, refluxing benzene (step (b)) to provide indenobenzopyran 4d.See, Shapiro, S. L.; Geiger, K.; Youlus, J.; Freedman, L. Indandiones AModified Dieckman Reaction J. Org. Chem. 1961, 26, 3580-3582. In anotheraspect, indenobenzopyran 4d was prepared by a novel, one-pot two-stepmethod, without isolation of intermediate 3d, which resulted in animproved yield (86%) compared to the previously reported synthesis yield(31%). See, Palior. M.; Worther, H.; Meller, A. Some reactions of2-aryl-1,3indandiones Monatsh Chem. 1961, 92, 1037-1047.

In another illustrative embodiment, substitutedbenz[d]indeno[1,2-b]pyran-5,11-diones 4 are prepared as shown in Scheme2. Treatment of optionally substituted phthalide 2 withN-bromosuccinimde in carbon tetrachloride/benzene under reflux (step(a)) affords brominated phthalide 2e. Treating brominated phthalide 2ewith aqueous acidic conditions under reflux (step (b)) affordshydroxylated phthalide 2f. Condensation of hydroxylated phthalide 2fwith optionally substituted phthalide 2 in a solution comprisingmethanol at room temperature, and an organic base, such as sodiummethoxide (step (c)), followed by reflux under acidic conditions (step(d)) affords optionally substituted indenobenzopyrans 4, where R^(a) andR^(d) are as defined herein.

In another illustrative embodiment, a novel, one-pot two-step processfor preparing substituted benz[d]indeno[1,2-b]pyran-5,11-diones 4 isdescribed. As shown in Scheme 3, condensation of optionally substituted2-carboxybenzaldehydes 1 and optionally substituted phthalides 2 inmethanol/ethyl acetate with sodium methoxide (step (a)) generatesintermediates 3, which are cyclized without isolation in acidified,refluxing benzene or via dicyclohexylcarbodiimide anddimethylaminopyridine (step (b)) to provide optionally substitutedindenobenzopyrans 4, where R^(a) and R^(d) are as defined herein.

In another illustrative embodiment, indenoisoquinoline compounds offormula I are prepared as outlined in Scheme 4. Treatment of theindenobenzopyrans 4 in chloroform with a primary amine of the formulaR⁶—(CH₂)_(m)—NH₂ (step (a)), where R⁶ and m are as defined herein,results in the formation of the corresponding indenoisoquinolines 5,where R^(a), R^(d), m, and R⁶ are as defined herein.

It is appreciated that although chloroform at room temperature willsuffice as the solvent for most primary amines, when a primary aminesuch as a mono-Boc-protected diamine, for example, is used to form thelactam from a benz[d]indeno[1,2-b]pyran-5,11-dione 4, chloroform atreflux may be used as the solvent. It is further appreciated that anindenoisoquinoline compound 5 for which the integer m is not 0, andwherein R⁶ is halo, azido, or cyano, for example, may be furtherelaborated through displacement of the halo, azido, or cyanofunctionality, respectively, with a variety of nucleophiles.Illustratively, treatment of indenobenzopyran 4 in chloroform with3-(bromo)propylamine (step (a)), i.e., a primary amine where R⁶ and m inthe formula R⁶—(CH₂)_(m)—NH₂ are bromo and 3, respectively, results inthe formation of the correspondingN-(3-bromo-1-propyl)indenoisoquinoline 5, which compound can be treatedwith sodium azide or sodium cyanide in DMSO or with primary andsecondary amines, such as ethanolamine, imidazole, N,N-dimethylamine,morpholine, piperazine, and the like, in refluxing dioxane, withconcomitant displacement of bromide ion. AnN-(3-cyano-1-propyl)indenoisoquinoline 5 may be converted to a varietyof carboxylic acid derivatives, including, for example, esters, amides,acid chlorides, and the like.

In another illustrative embodiment, indenoisoquinoline compounds offormula I are prepared as outlined in Scheme 5. Condensation ofoptionally substituted homophthalic anhydrides 6 with optionallysubstituted Schiff bases 7, where R^(a), R^(d), m, and R⁶ are as definedherein, generates carboxylic acids 8 (step (a)), for which the indicatedcis stereochemical relationship is based on the observed couplingconstant of ˜6 Hz for the two methine protons. (Carboxylic acids 9, witha trans stereochemical relationship, would be expected to display acoupling constant on the order of ˜10-12 Hz for the two methineprotons.) Subjecting carboxylic acids 8 to oxidative Friedel-Crafts ringclosure with thionyl chloride and aluminum chloride (step (b)) providesindenoisoquinolines 5, where R^(a), R^(d), m, and R⁶ are as definedherein.

In another illustrative embodiment, indenoisoquinoline compounds 10a-10oare described, where the various aspects and embodiments of m and R⁶ areas described herein, and R^(a) and R^(d) are as indicated in thefollowing table:

(I)

Compound R^(a) R^(d) 10a 2,3-(MeO)₂ 8,9-(MeO)₂ 10b 2,3-(MeO)₂8,9-(OCH₂O) 10c 2,3-(MeO)₂ 7,8,9-(MeO)₃ 10d 2,3-(OCH₂O) 8,9-(MeO)₂ 10e2,3-(OCH₂O) 8,9-(OCH₂O) 10f 2,3-(OCH₂O) 7,8,9-(MeO)₃ 10g 1,2,3-(MeO)₃8,9-(MeO)₂ 10h 1,2,3-(MeO)₃ 8,9-(OCH₂O) 10i 1,2,3-(MeO)₃ 7,8,9-(MeO)₃10j 1,4-(MeO)₂ 8,9-(MeO)₂ 10k 1,4-(MeO)₂ 8,9-(OCH₂O) 10l 1,4-(MeO)₂7,8,9-(MeO)₃ 10m 2,3,4-(MeO)₃ 8,9-(MeO)₂ 10n 2,3,4-(MeO)₃ 8,9-(OCH₂O)10o 2,3,4-(MeO)₃ 7,8,9-(MeO)₃

In another illustrative embodiment, novel compounds of formula II aredescribed

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein:

R^(a), R^(d), R^(a′), and R^(d′) each independently represent 4substituents, all of which are independently selected from the groupconsisting of hydrogen, halo, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, cyano, nitro, optionally substitutedalkylthio, optionally substituted alkylsulfonyl, carboxylic acid andderivatives thereof, and sulfonic acid and derivatives thereof; or where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle;and

X is a divalent linker comprising one or more divalent radicals selectedfrom —(CR¹R²), —(NR¹)— and —O—, where R¹ and R² are independentlyselected in each occurrence from hydrogen, alkyl, and acyl, providingthat the divalent linker does not include —O—O—. In one aspect, ifpresent, each divalent —(NR¹)— and —O— is separated by at least onedivalent radical (—CR¹R²)—. In another aspect, each R¹ and R² ishydrogen.

In another illustrative embodiment, X is a group having the generalstructure —(CH₂)_(n)—[(CH₂)_(x)—NR¹—(CH₂)_(y)]_(z)—(NR²)_(p)—(CH₂)_(q)—,where n is 0 or 1, x and y are integers independently ranging from 1 toabout 4, z is an integer ranging from 1 to about 4, p is 0 or 1, q is 0or an integer ranging from 1 to about 2, and where R¹ and R² areindependently selected in each instance from hydrogen, methyl,t-butyloxycarbonyl, benzyloxycarbonyl, and fluorenylmethoxycabonyl, orR¹ and R² and, any adjacent R² together with the attached nitrogens forma heterocycle.

In one illustrative embodiment of the compounds of formula II, R^(a) andR^(a′) independently represent one or more substituents selected fromoptionally substituted alkoxy. In one aspect, R^(a) and R^(a′)independently represent at least two adjacent substituents takentogether to form alkylenedioxy. In another embodiment, R^(a) and R^(a′)independently represent one or more substituents selected from halo,hydroxy, amino, alkyl and dialkylamino, nitroso, nitro, hydroxylamino,alkoxylamino, and cyano. In another embodiment of the compounds offormula II, R^(d) and R^(d′) independently represent one or moresubstituents selected from optionally substituted alkoxy. In one aspect,R^(d) and R^(d′) independently represent at least two adjacentsubstituents taken together to form alkylenedioxy. In anotherembodiment, R^(d) and R^(d′) independently represent one or moresubstituents selected from halo, amino, alkyl and dialkylamino, nitroso,nitro, and cyano.

In another illustrative embodiment of the compounds of formula II, n, p,and q are 0, and z is 2, 3, or 4. In another aspect, n and p are 1, z is1, and q is 2. In one aspect, R^(a) and R^(a′) independently representone or more substituents selected from optionally substituted alkoxy. Inanother aspect, R^(a) and R^(a′) independently represent at least twoadjacent substituents taken together to form alkylenedioxy. In anotheraspect, R^(a) and R^(a′) independently represent one or moresubstituents selected from halo, hydroxy, amino, alkyl and dialkylamino,nitroso, nitro, hydroxylamino, alkoxylamino, and cyano. In anotheraspect, R^(d) and R^(d′) independently represent one or moresubstituents selected from optionally substituted alkoxy. In anotheraspect, R^(d) and R^(d′) independently represent at least two adjacentsubstituents taken together to form alkylenedioxy. In another aspect,R^(d) and R^(d′) independently represent one or more substituentsselected from halo, amino, alkyl and dialkylamino, nitroso, and cyano.

In another illustrative embodiment, bisindenoisoquinoline compounds12-17 are described. These compounds were prepared by the processesdescribed herein comprising the steps of preparing and aminolyzing, witha suitable polyamine, a benz[d]indeno[1,2-b]pyran-5,11-dione 4 asdescribed herein.

(II)

Cpd R^(a) R^(a′) R^(d) R^(d′) —X— 12a H H H H CH₂NHCH₂ 12b H H H HCH₂CH₂NHCH₂ 12c H H H H CH₂CH₂NHCH₂CH₂ I2d H H H H CH₂CH₂N(CH₃)CH₂CH₂12e H H H H CH₂CH₂NHCH₂CH₂CH₂ 12f H H H H CH₂NH(CH₂)₂NHCH₂ 12g H H H HCH₂NH(CH₂)₃NHCH₂ 12h H H H H CH₂CH₂NH(CH₂)₂NHCH₂CH₂ 12i H H H HCH₂CH₂N(CH₂CH₂)₂NCH₂CH₂ 12j H H H H CH₂CH₂NH(CH₂)₃NHCH₂CH₂ 12k H H H HCH₂CH₂NH(CH₂)₄NHCH₂CH₂ 12l H H H H CH₂NH(CH₂)₂NH(CH₂)₂NHCH₂ 12m H H H HCH₂NH(CH₂)₂NH(CH₂)₂NHCH₂NHCH₂ 13a H H H H CH₂NBoc(CH₂)₃NBocCH₂ 13b H H HH CH₂CH₂NBoc(CH₂)₂NBocCH₂CH₂ 13c H H H H CH₂CH₂NBoc(CH₂)₃NBocCH₂CH₂ 13dH H H H CH₂CH₂NBoc(CH₂)₄NBocCH₂CH₂ 13e H H H HCH₂NBoc(CH₂)₂NBoc(CH₂)₂NBocCH₂ 13f H H H HCH₂NBoc(CH₂)₂NBoc(CH₂)₂NBoc(CH₂)₂NBocCH₂ 14a H H H H CH₂CH₂NHCH₂•TFA 14bH H H H CH₂CH₂NHCH₂CH₂CH₂•HCl 14c H H H H CH₂NH(CH₂)₂NHCH₂•2 TFA 14d H HH H CH₂NH(CH₂)₃NHCH₂•2 TFA 14e H H H H CH₂CH₂NH(CH₂)₂NHCH₂CH₂•2 TFA 14fH H H H CH₂CH₂N(CH₂CH₂)₂NCH₂CH₂•2 TFA 14g H H H HCH₂CH₂NH(CH₂)₃NHCH₂CH₂•2 TFA 14h H H H H CH₂CH₂NH(CH₂)₄NHCH₂CH₂•2 TFA14i H H H H CH₂NH(CH₂)₂NH(CH₂)₂NHCH₂•3 TFA 14j H H H HCH₂NH(CH₂)₂NH(CH₂)₂NHCH₂NHCH₂•4 TFA 14k 2,3-(MeO)₂ 2,3-(MeO)₂ H HCH₂NH(CH₂)₃NHCH₂•2 TFA 14l 2,3-(MeO)₂ 2,3-(MeO)₂ H HCH₂CH₂NH(CH₂)₃NHCH₂CH₂•2 TFA 14m 3-NO₂ 3-NO₂ H H CH₂NH(CH₂)₃NHCH₂•2 TFA14n 3-NO₂ 3-NO₂ H H CH₂CH₂NH(CH₂)₃NHCH₂CH₂•2 TFA 15a 2,3-(MeO)₂ H8,9-OCH₂O H CH₂CH₂NH(CH₂)₃NHCH₂CH₂ 16a 2,3-(MeO)₂ H 8,9-OCH₂O HCH₂CH₂NBoc(CH₂)₃NBocCH₂CH₂ 17a 2,3-(MeO)₂ H 8,9-OCH₂O HCH₂CH₂NH(CH₂)₃NHCH₂CH₂•2 TFA

It is appreciated that compounds 12-17 may be chemically more stablethan camptothecin, owing, at least in part, to the absence of thelactone ring. See, (a) Jaxel, C.; Kohn, K. W.; Wani, M. C.; Pornmier. Y.Structure-Activity Study of the Actions of Camptothecin Derivatives onMammalian Topoisomerase 1: Evidence for a Specific Receptor Site and aRelation to Antitumor Activity Cancer. Rev. 1989, 49, 1465-1469. (b)Minanri, H.; Beijnen, J. H.; Verweij, J.; Ratain, M. J. Limited SamplingModel for the Area under the Concentration Time Curve of Total TopotecanClin. Cancer Res. 1996, 2, 43.46. (c) Danks, M. K.; Pawlik, C. A.;Whipple, D. O.; Wolverton, J. S. Intermittant Exposure ofMedulloblastoma Cells to Topotecan Produces Growth Inhibition equivalentto Continuous Exposure Curr. Topics Med. Chem. 1997, 3, 1731-1738. (d)Haas. N. B.; LaCreta, F. P.; Walczak, J.; Nudes, G. R.; Brennan, J. M.;Ozols, R. F.; O'Dwyer, P. J. Phasel/Pharmaco-kinetic Study of Topotecanby 24-Hour Continuous Infusion Weekly Cancer Res. 1994, 54, 1220-1226,the disclosures of which are incorporated herein by reference. It isfurther appreciated that compounds 1247 may be efficacious againstvarious types of human cancers. It is also appreciated that compounds12-17 may have unique DNA binding site selectivities relative tocamptothecin.

In another illustrative embodiment, symmetrical bisindenoisoquinolinecompounds of formula II are prepared as outlined in Scheme 6. Treatmentof the indenobenzopyrans 4 in refluxing chloroform with a polyamine ofthe formulaNH₂—(CH₂)_(n)—[(CH₂)_(x)—NR¹—(CH₂)_(y)]_(z)—(NR²)_(p)—(CH₂)_(q)—NH₂ 11(step (a)), where R¹, R², n, x, y, z, p, and q are as defined herein,results in the formation of the corresponding bisindenoisoquinolines 12,where R^(a), R^(d), R^(a′), R^(d′), and X are as defined herein. Ifnecessary or desired, bisindenoisoquinolines 12 are converted to theirrespective t-butyloxycarbonyl (Boc-) derivatives 13 upon treatment withBoc anhydride and triethylamine (step (b)), then purified and treatedwith trifluoroacetic acid or hydrochloric acid (step (c)) to produce thecorresponding TFA or HCl salt 14.

In another illustrative embodiment, unsymmetrical bisindenoisoquinolinesof formula II are prepared as outlined in Scheme 7. Treatment of theindenobenzopyrans 4 with a polyamine of the formulaNH₂—(CH₂)_(n)—[(CH₂)_(x)—NR¹—(CH₂)_(y)]_(z)—(NR²)_(p)—(CH₂)_(q)—NH₂ 11(step (a)), where R¹, R², n, x, y, z, p, and q are as defined herein,results in the formation of the correspondingpolyaminoindenoisoquinoline A, where R^(a), R^(d), and X are as definedherein. Subsequent condensation of polyaminoindenoisoquinoline A withindenobenzopyran 4d (step (b)) results in the formation of thecorresponding unsymmetrical bisindenoisoquinolines 15. If necessary ordesired, unsymmetrical bisindenoisoquinolines 15 are converted to theirrespective t-butyloxycarbonyl (Boc-) derivatives 16 upon treatment withBoc anhydride and triethylamine (step (c)), then purified and treatedwith trifluoroacetic acid or hydrochloric acid (step (d)) to produce thecorresponding TFA or HCl salt 17.

It is appreciated that bisindenoisoquinolines 12 and 15 may be convertedto other acyl derivatives, including urethane derivatives such as, forexample, benzyloxycarbonyl or fluorenylmethoxycarbonyl derivatives, thenpurified and deprotected via hydrogenolysis or treatment withpiperidine, respectively.

The indenoisoquinoline and bisindenoisoquinoline compounds describedherein may also form hydrates and solvates. Hydrates may be formedspontaneously upon exposure to ambient conditions where the humidity issufficient to hydrate the compounds. In addition, hydrates may be formedwith more specificity by exposing the compounds described herein toparticular humidity conditions. Hydrates may also be formed with bydissolving or suspending the compounds in media containing apredetermined amount of water and evaporating, lyophilizing, orotherwise concentrating such solutions in a manner to give a hydrateform of the compounds described herein. Solvates of theindenoisoquinolinium and bisindenoisoquinolinium compounds describedherein may also be formed by dissolving or suspending the compounds in asolvent that is capable of forming a complex with the compound, andsubsequently evaporating or otherwise concentrating such solutions in amanner to give a solvate form of the compounds described herein.Solvents capable of forming solvates may include alcohols, such asethanol, butanol, and the like. It is appreciated that both hydrates andsolvates of the compounds described herein may have a predeterminedstoichiometry. Such stoichiometry may be evaluated by conventionalanalytical techniques, including X-ray diffraction, melting analysis,and the like.

The compounds described herein show antineoplastic activity using theCOMPARE screening methodology, demonstrating that they areantineoplastic agents useful in treating human cancers. The compoundsdescribed herein are inhibitors of topoisomerase I (top 1), and inparticular may be inhibitors of the top 1-catalyzed DNA religationreaction. Such inhibition may account for the antiproliferative activityagainst cancer cells that compounds described herein show in vitro. Thecompounds described herein may form ternary complexes consisting of thecompound, DNA, and the top1 enzyme. Without being bound by theory, it isbelieved that the compounds described herein may be operating as top1poisons, which inhibit the top1 enzyme catalyzed DNA cleavage reaction.It is further appreciated that the compounds described herein may havelonger in vitro and in vivo activity than conventional treatments if theformation of the ternary complexes are not reversible or rapidlyreversible.

Therefore, some of the growth inhibition demonstrated through COMPAREtesting may occur through that mechanism of action, inhibition oftopoisomerase I. However, it is appreciated that compounds showingsurprisingly potent cell growth inhibition, even though their inhibitoryeffects on topoisomerase I are relatively small in comparison to otheragents tested, may cause inhibition of cell growth, at least in part,through another mechanism of action in addition to or instead ofinhibition of topoisomerase I.

Also described herein are pharmaceutical compositions and formulationscomprising a therapeutically effective amount of one or moreindenoisoquinoline or bisindenoisoquinoline compounds for treating apatient having cancer. It is appreciated that mixtures of certainindenoisoquinoline or bisindenoisoquinoline compounds may beadministered. Such pharmaceutical compositions may also include one ormore diluents, carriers, and/or excipients. As used herein, an effectiveamount of the indenoisoquinoline or bisindenoisoquinoline compound isdefined as the amount of the compound which, upon administration to apatient, inhibits growth of cancer cells, kills malignant cells, reducesthe volume or size of the tumors, and/or eliminates the tumor entirelyin the treated patient. It is to be understood that treated patientsinclude humans and other mammals.

As used herein, the term “therapeutically effective amount” refers tothe amount to be administered to a patient, and may be based on bodysurface area, patient weight, and/or patient condition. In addition, itis appreciated that there is an interrelationship of dosages determinedfor humans and those dosages determined for animals, including testanimals (illustratively based on milligrams per meter squared of bodysurface) as described by Freireich, E. J., et al., Cancer Chemother.Rep. 1966, 50 (4), 219, the disclosure of which is incorporated hereinby reference. Body surface area may be approximately determined frompatient height and weight (see, e.g., Scientific Tables, GeigyPharmaceuticals, Ardley, New York, pages 537-538 (1970)). Atherapeutically effective amount of the indenoisoquinoline andbisindenoisoquinoline compounds described herein may be defined as anyamount useful for inhibiting the growth of (or killing) a population ofmalignant cells or cancer cells, such as may be found in a patient inneed of relief from such cancer or malignancy. Typically, such effectiveamounts range from about 5 mg/kg to about 500 mg/kg, from about 5 mg/kgto about 250 mg/kg, and/or from about 5 mg/kg to about 150 mg/kg ofindenoisoquinoline compounds per patient body weight. It is appreciatedthat effective doses may also vary depending on the route ofadministration, optional excipient usage, and the possibility ofco-usage of the indenoisoquinoline compounds with other conventional andnon-conventional therapeutic treatments, including other anti-tumoragents, radiation therapy, and the like.

The indenoisoquinoline and bisindenoisoquinoline compounds may beadministered in a variety of pharmaceutical formulations, includingconventional pharmaceutical formulations. The indenoisoquinolinecompounds, and formulated variants thereof, may also be delivered by avariety of administration routes, including conventional deliveryroutes. In one embodiment, the indenoisoquinoline compounds, andformulated variants thereof, are delivered via a parenteral route,including subcutaneously, intraperitoneally, intramuscularly, andintravenously. Examples of parenteral dosage forms and formulationsinclude aqueous solutions of the indenoisoquinoline compounds inisotonic saline, 5% glucose or other conventional pharmaceuticallyacceptable liquid carrier. In one aspect, the one or moreindenoisoquinoline compounds are dissolved in a saline solutioncontaining 5% dimethyl sulfoxide and 10% Cremphor EL (Sigma ChemicalCompany). Additional solubilizing agents such as cyclodextrins, whichcan form specific, more soluble complexes with the indenoisoquinolinecompounds described herein, or other conventional solubilizing agentscan be included as pharmaceutical excipients for delivery of thecompounds.

In another embodiment, the indenoisoquinoline compounds,bisindenoisoquinoline compounds, and formulated variants thereof, aredelivered via oral administration, such as in a capsule, a gel seal, atablet, and the like. Capsules may comprise any conventionalpharmaceutically acceptable material including gelatin and/or cellulosederivatives. Tablets may be formulated by conventional procedures,including by compressing mixtures of the indenoisoquinoline compounds,solid carriers, lubricants, disintegrants, and other conventionalingredients for solid dosage forms, such as starches, sugars, bentonite,and the like. The compounds described herein may also be administered ina form of a hard shell tablet or capsule containing, for example,lactose or mannitol as a binder, and conventional fillers and tabletingagents. Solid dosage forms described herein and useful for deliveringthe indenoisoquinoline compounds also include sustained releaseformulations, such as tablets, caplets, pills, capsules, and the likethat include an enteric coating that may delay the release of theindenoisoquinoline compounds until the formulation has passed into theintestinal tract.

The following exemplary embodiments are included herein to furtherillustrate the invention. These exemplary embodiments are not intendedand should not be interpreted to limit the scope of the invention in anyway. It is to be understood that numerous variations of these exemplaryembodiments are contemplated herein.

Compound Examples

Melting points were determined in capillary tubes and are uncorrected.Infrared spectra were obtained using CHCl₃ as the solvent unlessotherwise specified. Except where noted, 300 MHz ¹H NMR spectra wereobtained using CDCl₃ as solvent and the solvent peak as internalstandard. Mass spectra were determined by electrospray massspectrometry. Microanalyses were performed at the Purdue UniversityMicroanalysis Laboratory. Reactions were generally monitored byanalytical thin-layer chromatography using Baker-flex silica gel IB2-Fplates or flexible sheets, visualized with short wavelength UV light.Silica gel flash chromatography was performed using 230-400 mesh silicagel.

A representative procedure for the one-pot synthesis of anindenobenzopyran 4 is described herein forbenz[d]indeno[1,2-b]pyran-5,11-dione 4d. It is understood that otherindenobenzopyrans, including compounds 4a-4s, may be prepared accordingto this representative example. In addition, a representative procedurefor the synthesis of an indenoisoquinoline 5 from indenobenzopyran 4 anda primary amine is described herein for5,6-dihydro-6-(2-morpholinyl-1-ethyl)-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline5i. It is understood that other indenoisoquinolines, including compounds5a-5k, may be prepared from this representative example. Arepresentative procedure for the synthesis of an indenoisoquinoline 5 byaminolysis of6-(3-bromo-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline5y is also described herein, by which procedure indenoisoquinolines 51,and 5o-5x were prepared. In addition, a representative procedure for thesynthesis of an indenoisoquinoline 5 from indenobenzopyran 4d and amono-Boc-protected primary amine is described herein, by which proceduremono-Boc-protected indenoisoquinolines 5aa-5ac, and 5af-5ak wereprepared for generation of the corresponding HCl salts 5al-5an and5aq-5ay. Syntheses of indenoisoquinolines 5aw-5az from indenobenzopyran4d and a series of aminopyridine derivatives are also described herein.In addition, a representative procedure is described herein for thesynthesis of an indenoisoquinoline 5 by i) condensing substitutedhomophthalic anhydrides 6 with Schiff bases 7 and ii) subjecting theresulting carboxylic acids 8 to oxidative Friedel-Crafts ring closure,by which procedure indenoisoquinolines 5bb-5bc were prepared. It isunderstood that other indenoisoquinolines, including compounds 5ba, 5bd,and 5be, may be prepared according to this representative example.Indenoisoquinolines 5bf-5bs were prepared fromN-haloalkylindenoisoquinolines 5bb, 5bd and 5be by the aminolysisprocedure described herein. Also described herein are syntheses ofbisindenoisoquinolines 12-17 from indenobenzopyrans 4 and a variety ofpolyamines 11.

Benz[d]indeno[1,2-b]pyran-5,11-dione (4d). Sodium methoxide (40 mL of a4 M methanolic solution) was added to a solution of2-carboxybenzaldehyde 1d (1.000 g, 7.455 mmol) and phthalide 2d (1.119g, 7.455 mmol) in ethyl acetate (20 mL). The solution was heated at 65°C. for 18 h, concentrated, and acidified with coned HCl. The resultingmixture was diluted with benzene (125 mL), TsOH (100 mg) was added, andthe solution was heated for 7 h at reflux in a flask affixed with aDean-Stark trap. The solution was cooled to room temperature,concentrated, diluted with CHCl₃ (150 mL), and washed with sat NaHCO₃(3×50 mL) and sat NaCl (50 mL). The organic layer was dried over sodiumsulfate and concentrated to provide indenobenzopyran 4d as an orangesolid (1.583 g, 86%): mp 258-259° C. (published mp 257° C.). ¹H NMR(CDCl₃) δ 8.40 (d, J=8.56 Hz, 1H), 8.32 (d, J=7.93 Hz, 1H), 7.84-7.79(m, 1H), 7.61-7.39 (m, 5H). Additional details regarding the synthesisof compound 8 are found in Pailer et al., Monatsh Chem., 92:1037-47(1961), the synthetic disclosure of which is incorporated herein byreference.

5,6-Dihydro-6-(2-morpholinyl-1-ethyl)-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5i). 4-(2-Aminoethyl)morpholine (0.133 g, 1.023 mmol) was added to asolution of 3-nitrobenz[d]indeno[1,2-b]pyran-5,11-dione 4c (0.100 g,0.341 mmol) in CHCl₃ (30 mL). The solution was allowed to stir at roomtemperature for 16 h, diluted with CHCl₃ (110 mL) and washed with H₂O(3×30 mL) and sat NaCl (30 mL). The organic layer was dried over sodiumsulfate, filtered, and concentrated to provide indenoisoquinoline 9i asa crude solid. The solid was purified by flash column chromatography(SiO₂/CHCl₃ to 7% MeOH/CHCl₃) to provide indenoisoquinoline 5i as anorange solid (0.138 g, 100%): mp 257-259° C. IR (film) 1670, 1613, 1505,1330, and 1078 cm⁻¹; ¹H NMR (CDCl₃) δ 9.20 (s, 1H), 8.89 (d, J=Hz, 1H),8.52 (d, J=Hz, 1H), 7.80-7.72 (m, 2H), 7.54 (m, 2H), 4.73 (m, 2H), 3.72(bs, 4H), 2.83 (m, 2H), 2.62 (bs, 4H); ESIMS m/z (rel intensity) 406(MH⁺, 100). Anal. Calcd for C₂₂H₁₉N₃O₃: C, 65.18; H, 4.72; N, 10.37.Found: C, 65.27; H, 4.74; N, 10.20.

General Procedure for the Synthesis of Indenoisoquinolines 5l and 5o-5xfrom6-(3-bromo-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5y). A mixture of6-(3-bromo-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5y) (0.500 g, 1.06 mmol), amine (2.11 mmol), and anhydrous K₂CO₃ (0.584g, 4.23 mmol) in anhydrous 1,4-dioxane (30 mL) was heated at 100° C. for4 h. The reaction mixture was cooled and then concentrated. The residuewas diluted with water (50 mL), extracted with CHCl₃ (2×50 mL), washedwith 1% aq HCl (50 mL), water (50 mL), sat NaCl (50 mL), and dried overNa₂SO₄. The crude product was purified by flash column chromatography(SiO₂), eluting with a 0-5% gradient of methanol in chloroform, toprovide the pure indenoisoquinoline.

3-(Imidazolyl-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (51). The desired analogue was obtained as a dark purplesolid (245 mg, 63%): mp 316-318° C. ¹H NMR(CDCl₃) δ 8.01 (s, 1H), 7.63(s, 1H), 7.60 (s, 1H), 7.14 (s, 1H), 7.04 (s, 2H), 6.40 (s, 1H), 6.07(s, 2H), 4.45 (t, J=5.8 Hz, 2H), 4.20 (t, J=6.6 Hz, 2H), 4.03 (s, 3H),3.98 (s, 3H), 2.33 (t, J=6.9 Hz, 2H); ESIMS m/z (rel intensity) 460(MH⁺, 100). Anal. (C₂₅H₂₁N₃O₆.0.2H₂O) C, H, N. The hydrochloride saltwas formed by dissolving the product in chloroform (50 mL) and ananhydrous solution of 2 M HCl in diethyl ether (15 mL, 30.0 mmol) wasadded at 0° C. The reaction mixture was stirred at room temperature for6 h and the precipitated product was filtered and washed with chloroform(50 mL), methanol (20 mL), and dried over P₂O₅ for 24 h to afford theproduct as a dark purple solid (170 mg, 79%): mp 270-272° C. ¹H NMR(DMSO-d₆-CD₃OD, 2:1) δ 9.07 (s, 1H), 7.78 (s, 2H), 7.60 (s, 1H), 7.42(s, 1H), 7.14 (s, 1H), 6.96 (s, 1H), 6.13 (s, 2H), 4.41 (t, J=6.6 Hz,2H), 4.36 (t, J=7.3 Hz, 2H), 3.86 (s, 3H), 3.82 (s, 3H), 2.35 (t, J=6.1Hz, 2H); ESIMS m/z (rel intensity) 494 (MH⁺, 100). Anal. (C₂₅H₂₂N₃O₆Cl)C, H, N.

6-[3-Pyrazolyl-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5m).6-(3-Bromo-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5y) (0.2113 g, 0.448 mmol) was added to sodium hydride (86.8 mg of a60% suspension in mineral oil, 2.17 mmol) and pyrazole (0.1749 g, 2.57mmol) in DMF (50 mL) and the reaction mixture was heated at 60° C. for 4h. The reaction mixture was diluted with water (200 mL) and extractedwith chloroform (200 mL). The organic layer was washed with water (7×200mL) and concentrated. Benzene was added (2×30 mL) and the mixture wasconcentrated again. The residue was dissolved in chloroform (4 mL) anddiethyl ether (50 mL) was added. The precipitate was washed with diethylether (100 mL) and a dark red solid (118.5 mg, 57.6%) was obtained: mp262-264° C. (dec). IR. (film) 3462, 3104, 2918, 1693, 1640, 1557, 1495,1488, 1430, 1394, 1308, 1284, 1251, 1205, 868, 785, 769 cm⁻¹; ¹H NMR(DMSO-d₆) δ 7.97 (s, 1H), 7.65 (d, J=1.5 Hz, 1H), 7.61 (s, 1H), 6.57 (s,1H), 6.97 (s, 1H), 6.68 (s, 1H), 6.33 (s, 1H), 6.05 (s, 2H), 4.40 (m,4H), 4.01 (s, 3H), 3.96 (s, 3H), 2.45 (m, 2H); ESIMS m/z (rel intensity)460 (MH⁺, 100). Anal. (C₂₅H₂₁N₃O₆.0.75 H₂O) C, H, N.

6-(3-[2-(1,2,4)]-Triazolyl-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5n).6-(3-Bromo-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5y) (0.2538 g, 0.538 mmol) was added to sodium hydride (124.8 mg of a60% suspension in mineral oil, 3.12 mmol) and 1,2,4-triazole (0.2673 g,0.566 mol) in DMF (50 mL) and the reaction mixture was heated at 60° C.for 3 h. The reaction mixture was diluted with water (200 mL) and theprecipitate was separated by filtration and washed with water (50 mL).The precipitate was partially dissolved in methanol-chloroform 1:1 (200mL). Diethyl ether (100 mL) was added and the precipitate was separatedby filtration and washed with additional diethyl ether (100 mL) toprovide the product as the free base. The residue was dissolved intrifluoroacetic acid (2 mL) and hydrochloric acid (4 mL of a 2 Msolution in diethyl ether) was added, followed by more diethyl ether (30mL). The product was collected as a dark red solid (159.5 mg, 57%):mp>240° C. IR (KBr) 3429, 1694, 1647, 1553, 1500, 1487, 1431, 1394,1311, 1254, 1207, 1032, 928, 873, 800, 786, 722, 617 cm⁻¹; ¹H NMR(DMSO-d₆) δ 8.56 (s, 1H), 7.99 (s, 1H), 7.90 (s, 1H), 7.52 (s, 1H), 7.15(s, 1H), 7.10 (s, 1H), 6.19 (s, 2H), 4.44-4.38 (m, 4H), 3.90 (s, 3H),3.86 (s, 3H), 2.25 (m, 2H); ESIMS m/z (rel intensity) 461 (MH⁺, 53), 392(MH⁺—C₂N₃H₃, 100). High resolution ESIMS m/z (rel intensity) 461.1464(100, MH⁺) (calculated mass 461.1461).

6-(3-Thiazolylamino-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineDihydrochloride (5o). The product (213 mg, 41%) was dissolved inchloroform (50 mL) and treated with an anhydrous solution of 2 M HCl indiethyl ether (15 mL, 30.0 mmol) at 0° C. The reaction mixture wasstirred at room temperature for 6 h and the precipitated product wasfiltered and washed with chloroform (50 mL), methanol (10 mL), and driedover P₂O₅ to provide the desired analogue as a pale purple solid (140mg, 61%): mp 298-300° C. (deo). ¹H NMR (DMSO-d₆) δ 7.82 (s, 1H), 7.44(s, 1H), 7.38 (s, 1H), 7.04 (s, 1H), 6.18 (s, 2H), 4.42 (bs, 2H), 4.07(bs, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 3.76 (bs, 4H), 2.07 (bs, 2H);ESIMS m/z (rel intensity) 494 (MH⁺, 100). Anal. (C₂₅H₂₅N₃O₆SCl₂.0.6CHCl₃) C, H, N.

6-(3-piperazinyl-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineDihydrochloride (5p). The product (350 mg, 72%) was dissolved inchloroform and treated with 2 M HCl in diethyl ether (9.0 mL, 18.2 mmol)at room temperature to afford the desired analogue as a pale purplesolid (280 mg, 84%): mp 276-278 (dec). ¹H NMR (D₂O) δ 6.63 (bs, 1H),6.53 (s, 1H), 6.47 (bs, 1H), 6.18 (s, 1H), 5.91 (s, 2H), 3.90 (bs, 2H),3.51 (s, 3H), 3.46 (bs, 11H), 3.20 (bs, 2H), 2.02 (bs, 2H); ESIMS m/z(rel intensity) 478 (MH⁺, 100). Anal. (C₂₆H₂₉Cl₂N₃O₆.2.3H₂O) C, H, N.

3-[(Morpholinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5q). The product was isolated as a dark purple solid (0.220 g, 72%): mp290-292° C. ¹H NMR (CDCl₃) δ 7.98 (s, 1H), 7.59 (s, 1H), 7.36 (s, 1H),7.02 (s, 1H), 6.07 (s, 2H), 4.48 (t, J=7.39 Hz, 2H), 4.02 (s, 3H), 3.95(s, 3H), 3.76 (bs, 4H), 2.54 (bs, 6H), 2.01 (bs, 2H); ESIMS m/z (relintensity) 479 (MH⁺, 100). Anal. (C₂₆H₂₆N₂O₇.0.2H₂O) C, H, N.

3-[(Thiomorpholinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5r). The product was isolated as a dark purple solid (275 mg, 53%): rap306-308° C. ¹H NMR (CDCl₃) δ 7.80 (s, 1H), 7.60 (s, 1H), 7.33 (s, 1H),7.04 (s, 1H), 6.08 (s, 2H), 4.48 (t, J=6.4 Hz, 2H), 4.02 (s, 3H), 3.96(s, 3H), 2.84-2.78 (bs, 8H), 2.67 (bs, 2H), 2.09 (bs, 2H); ESIMS m/z(rel intensity) 495 (MH⁺, 100). Anal. (C₂₆H₂₆N₂O₆S.0.3 H₂O) C, H, N.

6-[3-(3-Hydroxypiperidinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5s). The product (220 mg, 0.45 mmol, 70%) was treatedwith 2 M HCl in diethyl ether (4.0 mL, 6.69 mmol) in chloroform at roomtemperature to afford the desired analogue as a purple solid (210 mg,89%): mp 288-290° C. ¹H NMR (D₂O) δ 6.54 (bs, 1H), 6.41 (s, 1H), 6.29(bs, 1H), 6.06 (s, 1H), 5.88 (s, 2H), 3.82 (bs, 2H), 3.45 (s, 3H), 3.37(bs, 7H), 3.15 (bs, 3H), 1.99 (bs, 4H), 1.68 (bs, 2H); ESIMS m/z (relintensity) 493 (MH⁺, 100). Anal. (C₂₇H₂₉ClN₂O₇.1.4H₂O) C, H, N.

3-[(1-Methylpiperazinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5t). The desired analogue was isolated as a dark purple solid (160 mg,51%): mp 254-256° C. ¹H NMR (CDCl₃) δ 7.99 (s, 1H), 7.60 (s, 1H), 7.30(s, 1H), 7.03 (s, 1H), 6.08 (s, 2H), 4.47 (t, J=6.0 Hz, 2H), 4.02 (s,3H), 3.96 (s, 3H), 2.55 (bs, 10H), 2.30 (s, 3H), 1.99 (bs, 2H); ESIMSm/z (rel intensity) 492 (MH⁺, 100). Anal. (C₂₇H₂₉N₃O₆.0.5 CHCl₃) C, H,N.

6-[3-(4-Aminopiperidinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineDihydrochloride (5u). The product (205 mg, 66%) was dissolved inchloroform (30 mL) and treated with 2 M HCl in diethyl ether (5.2 mL,10.40 mmol) at room temperature for 8 h. The precipitate was filteredand washed with chloroform (30 mL) to provide the desired analogue as adark purple solid (165 mg, 85%): mp 262-264° C. (dec). ¹H NMR (D₂O) δ6.62 (s, 1H), 6.50 (s, 1H), 6.44 (s, 1H), 6.17 (s, 1H), 5.92 (s, 2H),3.92 (bs, 2H), 3.64 (bs, 2H), 3.50 (s, 4H), 3.45 (s, 3H), 3.23 (bs, 2H),3.08 (bs, 2H), 2.25 (m, 2H), 2.06 (bs, 2H), 1.90 (m, 2H); ESIMS m/z (relintensity) 492 (MH⁺, 70).

6-(3-Homopiperazinyl-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineDihydrochloride (5v). The obtained product (390 mg, 0.66 mmol, 69%) wasdissolved in chloroform and treated with 2 M HCl in diethyl ether (10.0mL, 19.8 mmol) to afford the desired analogue as a purple solid (305 mg,82%): mp 264-266° C. (dec). ¹H NMR (D₂O) δ 6.71 (bs, 1H), 6.56 (bs, 2H),6.21 (bs, 1H), 5.92 (s, 2H), 3.98 (bs, 2H), 3.63-3.57 (bs, 6H), 3.55 (s,3H), 3.50 (s, 3H), 3.36-3.25 (bs, 4H), 2.19 (bs, 2H), 2.09 (bs, 2H);ESIMS m/z (rel intensity) 492 (MH⁴⁻, 100). Anal. (C₂₇H₃₁Cl₂N₃O₆.0.7H₂O)C, H, N.

3-[(1-Hydroxyethyl-piperazine)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5w). The desired analogue was isolated as a dark brown solid (258 mg,47%): mp 262-264° C. ¹H NMR (CDCl₃) δ 8.00 (s, 1H), 7.60 (s, 1H), 7.32(s, 1H), 7.04 (s, 1H), 6.06 (s, 2H), 4.52 (bs, 2H), 4.03 (s, 3H), 3.96(s, 3H), 3.22 (bs, 4H), 3.13 (bs, 6H), 2.84 (bs, 2H), 2.68 (bs, 2H),1.73 (bs, 4H), 1.63 (bs, 4H), 1.43 (s, 18H), 1.41 (s, 9H); ESIMS m/z(rel intensity) 522 (MH⁺, 100). Anal. (C₂₈H₃₁N₃O₇.0.8H₂O) C, H, N.

6-[(3-Morpholylethylamino)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5x). The desired analogue was isolated as a pale purple solid (245 mg,59%): mp 215-217° C. ¹H NMR (CDCl₃) δ 8.01 (s, 1H), 7.62 (s, 1H), 7.42(s, 1H), 7.05 (s, 1H), 6.06 (s, 2H), 4.52 (bs, 2H), 4.03 (s, 3H), 3.97(s, 3H), 3.70 (be, 4H), 2.81 (bs, 2H), 2.73 (bs, 2H), 2.53 (bs, 2H),2.46 (bs, 4H), 2.02 (bs, 2H); ESIMS m/z (rel intensity) 522 (MH⁺, 100).Anal. (C₂₈H₃₁N₃O₇.1.0 H₂O) C, H, N.

General Procedure for the Preparation of Mono-Boc-Protected Diamines.Boc₂O (0.500 g, 2.291 mmol) was dissolved in CHCl₃ (10 mL) and thesolution was added dropwise to a solution of diamine (11.45 mmol) inCHCl₃ (50 mL). The reaction mixture was allowed to stir at roomtemperature for 24 h, concentrated, and purified by flash columnchromatography (SiO_(r)), eluting with a solution of 1% Et₃N/10% MeOH inCHCl₃, to provide the mono-Boc protected diamine.(Mono-Boc-1,2-diaminoethane, mono-Boc-1,3-diaminopropane, andmono-Boc-1,4-diaminobutane were also prepared as described below.)

Mono-Boc-1,7-diaminoheptane The general procedure provided the desiredcompound as a colorless semisolid (0.473 g, 90%). ¹H NMR (CDCl₃) δ 4.52(bs, 1H), 3.12 (q, J=6.2 Hz, 2H), 2.70 (t, J=6.8 Hz, 2H), 1.43-1.23 (m,19H).

Mono-Boc-1,8-diaminooctane The general procedure provided the desiredcompound as a colorless semisolid (0.492 g, 88%). ¹H NMR (CDCl₃) δ 4.51(bs, 1H), 3.12 (q, J=6.5 Hz, 2H), 2.69 (t, J=6.8 Hz, 2H), 1.43-1.23 (m,21H).

Mono-Boc-1,9-diaminononane. The general procedure provided the desiredcompound as a colorless semisolid (0.125 g, 21%). ¹H NMR (CDCl₃) δ 4.50(bs, 1H), 3.12 (q, J=6.5 Hz, 2H), 2.70 (t, J=6.8 Hz, 2H), 1.44-1.22 (m,23H).

Mono-Boc-1,10-diaminodecane. The general procedure provided the desiredcompound as a colorless semisolid (0.192 g, 31%). ¹H NMR (CDCl₃) δ ¹HNMR (CDCl₃) δ 4.50 (bs, 1H), 3.13 (q, J=6.3 Hz, 2H), 2.71 (t, J=6.9 Hz,2H), 1.44-1.18 (m, 27H).

Mono-Boc-1,11-diaminoundecane. The general procedure provided thedesired compound as a colorless solid (0.555 g, 85%): mp 30-34° C. IR(film) 3370, 2919, 2851, 1687, and 1522 cm⁻¹; ¹H NMR (CDCl₃) δ 4.49 (bs,1H), 3.11 (q, J=6.5 Hz, 2H), 2.71 (t, J=6.8 Hz, 2H), 1.44-1.27 (m, 29H);ESIMS m/z (rel intensity) 287 (MH⁺, 100). Anal. (C₁₆H₃₄N₂O₂) C, H, N.

Mono-Boc-1,12-diaminododecane. The general procedure provided thedesired compound as a colorless semisolid (0.191 g, 28%). ¹H NMR (CDCl₃)δ 4.48 (bs, 1H), 3.11 (q, J=6.2 Hz, 2H), 2.76 (t, J=6.9 Hz, 2H),1.44-1.26 (m, 31H).

6-Amino-5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline (5z).Benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.150 g, 0.604 mmol) wastreated with hydrazine (0.255 g, 7.964 mmol) in CHCl₃ (50 mL) and thereaction mixture was heated at reflux for 16 h. The reaction mixture wasallowed to cool to room temperature, diluted with CHCl₃ (150 mL), andwashed with sat NaHCO₃ (2×50 mL). The solution was dried over sodiumsulfate and concentrated to provide a red-orange solid (0.120 g, 76%):mp 272-274° C. IR (film) 3448, 3305, 1686, 1663, 1610, 1507, 1312, 762cm⁻¹; ¹H NMR (CDCl₃) δ 8.54 (d, J=7.8 Hz, 1H), 8.51 (d, J=7.2 Hz, 1H),8.24 (d, J=7.4 Hz, 1H), 7.85 (m, 1H), 7.60-7.45 (m, 4H), 6.19 (s, 2H);EIMS m/z (rel intensity) 262 (M⁺, 100). Anal. (C₁₆H₁₀N₂O₂.0.25H₂O) C, H,N.

General Procedure for the Preparation of Mono-Boc-ProtectedIndenoisoquinolines. Mono-Boc protected diamine (2.054 mmol) was addedto a solution of benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.255 g,1.027 mmol) in CHCl₃ (100 mL). The reaction mixture was heated at refluxfor 24 h, concentrated, and purified by flash column chromatography(SiO₂), eluting with CHCl₃, to provide the mono-Boc-protectedindenoisoquinoline. (Mono-hoc-protected indenoisoquinolines 5aa, 5ab,and 5ac were also prepared as described below.)

6-(7′-tert-BOC-Aminoheptyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5af). The general procedure provided the desired compound as ayellow-orange solid (0.451 g, 95%): mp 112-116° C. IR (film) 3369, 1697,1664, 1503, and 1172 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.58 (d, J=8.1 Hz, 1H),8.23 (d, J=8.2 Hz, 1H), 7.84-7.79 (m, 1H), 7.71 (d, J=7.7 Hz, 1H),7.63-7.50 (m, 4H), 6.76 (m, 1H), 4.50 (t, J=7.4 Hz, 2H), 2.90 (q, J=6.2Hz, 2H), 1.77 (m, 2H), 1.46-1.28 (m, 17H); ESIMS m/z/z (rel intensity)483 (MNa⁺, 100). Anal. (C₂₈H₃₂N₂O₄) C, H, N.

6-(8′-tert-BOC-Aminooetyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5ag). The general procedure provided the desired compound as ayellow-orange solid (0.466 g, 97%): mp 140-143° C. IR (film) 3368, 2929,1698, 1665, 1504, and 1172 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.58 (d, J=7.9 Hz,1H), 8.23 (d, J=8.1 Hz, 1H), 7.84-7.78 (m, 1H), 7.71 (d, J=7.5 Hz, 1H),7.63-7.47 (m, 4H), 6.75 (m, 1H), 4.50 (t, J=7.4 Hz, 2H), 2.91 (q, J=6.6Hz, 2H), 1.78 (m, 2H), 1.46-1.26 (m, 19H); ESIMS m/z (rel intensity) 497(MNa⁺, 100). Anal. (C₂₉H₃₄N₂O₄) C, H, N.

6-(9′-tert-BOC-Aminononyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5ah). The general procedure provided the desired compound as an orangesolid (0.145 g, 77%): mp 91-95° C. IR (film) 3371, 2928, 1698, 1666,1504, and 1172 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.58 (d, J=8.0 Hz, 1H), 8.23 (d,J=7.4 Hz, 1H), 7.84-7.79 (m, 1H), 7.71 (d, J=7.4 Hz, 1H), 7.63-7.50 (m,4H), 6.74 (m, 1H), 4.50 (t, J=7.3 Hz, 2H), 2.91 (q, J=6.6 Hz, 2H), 1.78(m, 2H), 1.47-1.24 (m, 21H); ESIMS m/z (rel intensity) 511 (MNa⁺, 100).Anal. (C₃₀H₃₆N₂O₄) C, H, N.

6-(10′-tert-BOC-Aminodecyl)-5,6-dihydro-5,1-dioxo-11H-indeno[1,2-e]isoquinoline(5ai). The general procedure provided the desired compound as ayellow-orange solid (0.220 g, 78%): mp 135-137° C. IR (film) 3368, 2927,1698, 1666, 1504, and 1172 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.58 (d, J=7.9 Hz,1H), 8.23 (dd, J=8.1 Hz and 0.7 Hz, 1H), 7.84 (dt, J=7.2 Hz and 1.4 Hz,1H), 7.71 (d, J=7.5 Hz, 1H), 7.63-7.47 (m, 4H), 6.76 (m, 1H), 4.50 (t,J=7.4 Hz, 2H), 2.90 (q, J=6.5 Hz, 2H), 1.77 (m, 2H), 1.46-1.23 (m, 23H);ESIMS m/z (rel intensity) 525 (MNa⁺, 100). Anal. (C₃₁H₃₈N₂O₄) C, H, N.

6-(11′-tert-BOC-Aminoundecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5aj). The general procedure provided the desired compound as ayellow-orange solid (0.445 g, 86%): mp 111-114° C. IR (KBr) 3364, 2918,2850, 1678, 1660, 1534, 1505, and 758 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.58 (d,J=8.1 Hz, 1H), 8.23 (d, J=7.4 Hz, 1H), 7.84-7.79 (m, 1H), 7.71 (d, J=7.5Hz, 1H), 7.62-7.50 (m, 4H), 6.74 (m, 1H), 4.50 (t, J=7.4 Hz, 2H), 2.90(q, J=6.5 Hz, 2H), 1.78 (m, 2H), 1.46-1.22 (m, 25H); ESIMS m/z (relintensity) 539 (MNa⁺, 100). Anal. (C₃₂H₄₀N₂O₄) C, H, N.

6-(12′-tert-BOC-Aminododecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5ak). The general procedure provided the desired compound as ayellow-orange solid (0.177 g, 66%): mp 129-134° C. IR (film) 3369, 2926,1698, 1666, 1504, and 1172 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.58 (d, J=8.0 Hz,1H), 8.22 (d, J=7.4 Hz, 1H), 7.84-7.78 (m, 1H), 7.71 (d, J=7.5 Hz, 1H),7.62-7.50 (m, 4H), 6.74 (m, 1H), 4.50 (t, J=7.3 Hz, 2H), 2.90 (q, J=6.6Hz, 2H), 1.77 (m, 2H), 1.46-1.22 (m, 27H); ESIMS (rel intensity) 553(MNa⁺, 100). Anal. (C₃₃H₄₂N₂O₄) C, H, N.

6-(5-Aminopentyl)-5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinolineHydrochloride (5ao). Benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.100 g,0.403 mmol) was treated with 1,5-diaminopentane (0.206 g, 2.014 mmol) inCHCl₃ (40 mL) and the reaction mixture was heated at reflux for 16 h.The reaction mixture was allowed to cool to room temperature and washedwith water (3×15 mL). The solution was dried over sodium sulfate,filtered, and treated with 2 M HCl in Et₂O (5 mL). After 30 min, thereaction mixture was filtered and the filter pad was washed with CHCl₃(50 mL) and hexanes (50 mL) to provide an orange solid (0.122 g, 82%):mp 265-268° C. IR (film) 3432, 3077, 2856, 1707, 1635, 1611, 1549, and1504 cm⁻¹; ¹H NMR. (CDCl₃) δ 8.59 (d, J=8.1 Hz, 1H), 8.23 (d, J=8.1 Hz,1H), 7.85-7.80 (m, 3H), 7.74 (d; J=7.4 Hz, 1H), 7.63-7.51 (m, 4H), 4.52(t, J=7.3 Hz, 2H), 2.81 (m, 2H), 1.83 (m, 2H) 1.65-1.52 (m, 4H); ESIMSm/z (rel intensity) 333 (MH⁺, 100). Anal. (C₂₁H₂₁ClN₂O₂.0.75 H₂O) C, H,N.

6-(6-Aminohexyl)-5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinolineHydrochloride (5ap). Benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.100 g,0.403 mmol) was treated with 1,6-diaminohexane (0.234 g, 2.014 mmol) inCHCl₃ (40 mL) and the reaction mixture was heated at reflux for 16 h.The reaction mixture was allowed to cool to room temperature and washedwith water (3×25 mL). The solution was dried over sodium sulfate,filtered, and treated with 2 M HCl in Et₂O (5 mL). After 30 min, thereaction mixture was filtered and the filter pad was washed with CHCl₃(50 mL) and hexanes (50 mL) to provide an orange solid (0.125 g, 81%):mp 195° C. (dec). IR (film) 3435, 1660, 1630, 1610, and 1504 cm⁻¹; ¹HNMR (CDCl₃) δ 8.59 (d, J=7.8 Hz, 1H), 8.23 (d, J=8.1 Hz, 1H), 7.85-7.71(m, 4H), 7.61-7.51 (m, 4H), 4.52 (t, J=7.3 Hz, 2H), 2.78 (m, 2H), 1.79(m, 2H), 1.59-1.39 (m, 6H); ESIMS m/z (rel intensity) 347 (MH⁺, 100).Anal. (C₂₂H₂₃ClN₂O₂.0.5H₂O) C, H, N.

General Procedure for the Preparation of IndenoisoquinolineHydrochloride Salts. 3 M HCl in MeOH (10 mL) was slowly added to asolution of mono-Boc protected indenoisoquinoline (0.100 g, 0.188-0.217mmol) in CHCl₃ (50 mL) at room temperature. After 2 h, the reactionmixture was concentrated and the residue was triturated with Et₂O.Filtration of the obtained solid provided the indenoisoquinoline as ahydrochloride salt. (Indenoisoquinoline hydrochloride salts 5al, 5am,and 5an were also prepared as described below.)

6-(7-Aminoheptyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5aq). The general procedure provided the desired compoundas a yellow-orange solid (0.085 g, 99%): mp 228-231° C. IR (KBr) 3436,2931, 1702, 1650, 1611, 1549, 1504, and 759 cm⁻¹; ¹H NMR (DMSO-d₆) δ8.60 (d, J=8.1 Hz, 1H), 8.24 (d, J=8.2 Hz, 1H), 7.85-7.80 (m, 1H), 7.73(d, J=7.4 Hz, 1H), 7.63-7.49 (m, 6H), 4.53 (t, J=7.0 Hz, 2H), 2.78 (t,J=7.1 Hz, 2H), 1.80 (m, 2H), 1.55-1.35 (xn, 8H); ESIMS m/z (relintensity) 361 (MH⁺, 100). Anal. (C₂₃H₂₅ClN₂O₂.0.5H₂O) C, H, N.

6-(8-Aminooctyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5ar). The general procedure provided the desired compoundas an orange solid (0.083 g, 95%): mp 182-185° C. IR (KBr) 3436, 2930,1661, 1505, and 761 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.60 (d, J=8.5 Hz, 1H),8.24 (d, J=7.0 Hz, 1H), 7.86-7.81 (m, 1H), 7.73 (d, J=7.6 Hz, 1H),7.63-7.52 (m, 6H), 4.52 (t, J=7.9 Hz, 2H), 2.78 (t, J=7.3 Hz, 2H), 1.79(m, 2H), 1.50 (m, 4H), 1.31 (m, 6H); ESIMS m/z (rel intensity) 375 (MH⁺,100). Anal. (C₂₄H₂₇ClN₂O₂.0.75H₂O) C, H, N.

6-(9-Aminononyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5 as). The general procedure provided the desiredcompound as an orange solid (0.082 g, 94%): mp 204-207° C. IR (KBr)3435, 2927, 1702, 1662, 1610, 1549, 1504, 1427, and 759 cm⁻¹; ¹H NMR(DMSO-d₆) δ 8.60 (d, J=8.3 Hz, 1H), 8.24 (d, J=9.3 Hz, 1H), 7.83-7.81(m, 1H), 7.73 (d, J=7.8 Hz, 1H), 7.63-7.51 (m, 6H), 4.52 (t, J=8.3 Hz,2H), 2.78 (t, J=7.3 Hz, 2H), 1.79 (m, 2H), 1.51 (m, 4H), 1.28 (m, 8H);ESIMS m/z (rel intensity) 389 (MH⁺, 100). Anal. (C₂₅H₂₉ClN₂O₂.0.75H₂O)C, H, N.

6-(10-Aminodecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5at). The general procedure provided the desired compoundas an orange solid (0.087 g, 91%): mp 189-192° C. JR (KBr) 3443, 2925,2851, 1705, 1646, 1611, 1550, 1504, 1467, and 759 cm⁻¹; ¹H NMR (DMSO-d₆)δ 8.60 (d, J=7.9 Hz, 1H), 8.23 (d, 0.1=7.5 Hz, 1H), 7.83 (m, 1H), 7.73(d, J=8.0 Hz, 1H), 7.63-7.51 (m, 6H), 4.52 (t, J=7.4 Hz, 2H), 2.76 (m,2H), 1.79 (m, 2H), 1.49 (m, 4H), 1.27 (m, 10H); ESIMS m/z (relintensity) 403 (MH⁺, 100). Anal. (C₂₆H₃₁ClN₂O₂.0.5H₂O) C, H, N.

6-(11-Aminoundecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5au). The general procedure provided the desired compoundas an orange solid (0.085 g, 88%): mp 125-129° C. IR (KBr) 3436, 2922,2851, 1662, 1610, 1549, 1504, 1426, and 758 cm⁻¹; ¹H NMR (DMSO-d₆) δ8.60 (d, J=8.0 Hz, 1H), 8.23 (d, J=7.5 Hz, 1H), 7.85 (m, 1H), 7.72 (d,J=7.5 Hz, 1H), 7.63-7.51 (m, 6H), 4.51 (t, J=8.0 Hz, 2H), 2.77 (t, J=7.6Hz, 2H), 1.78 (m, 2H), 1.48 (m, 4H), 1.25 (m, 12H); ESIMS m/z (relintensity) 417 (MH⁺, 100). Anal. (C₂₆H₃₁ClN₂O₂.1H₂O) C, H, N.

6-(12-Aminododecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (5av). The general procedure provided the desired compoundas a yellow solid (0.087 g, 91%): mp 175-178° C. IR (KBr) 3435, 2927,2850, 1704, 1644, 1506, 1466, and 762 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.59 (d,J=7.8 Hz, 1H), 8.23 (d, J=7.5 Hz, 1H), 7.83 (m, 1H), 7.72 (d, J=7.8 Hz,1H), 7.63-7.51 (m, 6H), 4.51 (t, J=7.5 Hz, 2H), 2.76 (m, 2H), 1.78 (m,2H), 1.51 (m, 4H), 1.24 (m, 14H); ESIMS m/z (rel intensity) 431 (MH⁺,100). Anal. (C₂₈H₃₅ClN₂O₂.1.25H₂O) C, H, N.

5,6-Dihydro-5,11-dioxo-6-(2-pyridylmethyl)-11H-indeno[1,2-c]isoquinoline(5aw). 2-(Aminomethyl)pyridine (0.054 g, 0.504 mmol) was added to asolution of benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.100 g, 0.403mmol) in CHCl₃ (50 mL) and the reaction mixture was heated at reflux for16 h. The reaction mixture was allowed to cool to room temperature,washed with H₂O (3×25 mL), sat NaCl (25 mL), dried over sodium sulfate,and concentrated. The residue was washed with EtOAc, hexanes, and driedto provide a yellow solid (0.110 g, 81%): mp 240-242° C. IR (KBr) 1698,1655, 1618, 1501, 1427, and 755 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.62 (d, J=8.0Hz, 1H), 8.56 (d, J=4.9 Hz, 1H), 8.22 (d, J=8.1 Hz, 1H), 7.97 (dt, J=7.8Hz and 1.7 Hz, 1H), 7.89 (m, 1H), 7.61-7.37 (m, 7H), 5.91 (s, 2H); ESIMSm/z (rel intensity) 339 (MH⁺, 100). Anal. (C₂₂H₁₄N₂O₂) C, H, N.

5,6-Dihydro-5,11-dioxo-6-(3-pyridylmethyl)-11H-indeno[1,2-c]isoquinolineHydrochloride (5ax). 3-(Aminomethyl)pyridine (0.054 g, 0.504 mmol) wasadded to a solution of benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.100g, 0.403 mmol) in CHCl₃ (50 mL) and the reaction mixture was heated atreflux for 16 h. The reaction mixture was allowed to cool to roomtemperature, washed with H₂O (3×25 mL), sat NaCl (25 mL), dried oversodium sulfate, and concentrated. The residue was diluted with CHCl₃ (40mL), 3 M HCl in MeOH (10 mL) was added, and the reaction mixture wasallowed to stir at room temperature for 2 h. The reaction mixture wasconcentrated, and the residue was washed with CHCl₃ to provide a pinksolid (0.146 g, 97%): mp 274° C. (dec). IR (KBr) 2343, 2106, 1695, 1655,1610, 1551, 1501, and 754 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.98 (s, 1H), 8.78(d, J=5.2 Hz, 1H), 8.64 (d, J=8.1 Hz, 1H), 8.36 (d, J=9.1 Hz, 1H), 8.23(d, J=7.5 Hz, 1H), 7.90 (m, 2H), 7.59-7.39 (m, 5H), 5.89 (s, 2H); ESIMSm/z (rel intensity) 339 (MH⁺, 100). Anal. (C₂₂H₁₅ClN₂O₂) C, H, N.

5,6-Dihydro-5,11-dioxo-6-(2-pyridylethyl)-11H-indeno[1,2-c]isoquinolineHydrochloride (5ay), 2-(2-Aminoethyl)pyridine (0.098 g, 0.806 mmol) wasadded to a solution of benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.100g, 0.403 mmol) in CHCl₃ (50 mL) and the reaction mixture was heated atreflux for 16 h. The reaction mixture was allowed to cool to roomtemperature, washed with H₂O (3×25 mL), sat NaCl (25 mL), dried oversodium sulfate, and concentrated. The residue was diluted with CHCl₃ (40mL), 3 M HCl in MeOH (10 mL) was added, and the reaction mixture wasallowed to stir at room temperature for 2 h. The reaction mixture wasconcentrated, and the residue was washed with CHCl₃ to provide a yellowsolid (0.146 g, 93%): mp 240° C. (dec). IR (KBr) 2307, 1698, 1659, 1610,1548, 1504, 1429, and 760 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.78 (d, J=5.5 Hz,1H), 8.57 (d, J=8.0 Hz, 1H), 8.33 (m, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.97(d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.80 (m, 2H), 7.61-7.44 (m,4H), 4.92 (t, J=6.5 Hz, 2H), 3.59 (t, J=6.3 Hz, 2H); ESIMS m/z (relintensity) 353 (MH⁺, 100). Anal. (C₂₃H₁₇ClN₂O₂) C, H, N.

5,6-Dihydro-5,11-dioxo-6-(3-pyridylethyl)-11H-indeno[1,2-c]isoquinoline(5az). 3-(2-Aminoethyl)pyridine (0.172 g, 0.604 mmol) was added to asolution of benz[d]indeno[1,2-b]pyran-5,11-dione (4d) (0.100 g, 0.403mmol) in CHCl₃ (50 mL). Triethylamine (0.224 mL, 1.612 mmol) was addedand the reaction mixture was heated at reflux for 16 h. The reactionmixture was allowed to cool to room temperature, washed with H₂O (3×25mL), sat NaCl (25 mL), dried over sodium sulfate, and concentrated. Theobtained precipitate was washed with EtOAc, hexanes, and dried toprovide an orange solid (0.140 g, 99%): mp 220° C. (dec). IR (KBr) 1691,1660, 1609, 1549, 1504, 1424, and 765 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.90 (s,1H), 8.73 (d, J=. 5.6 Hz, 1H), 8.59 (d, J=8.1 Hz, 1H), 8.44 (d, J=7.9Hz, 1H), 8.10 (d, J=8.1 Hz, 1H), 7.88 (m, 3H), 7.61-7.48 (m, 4H), 4.87(t, J=6.7 Hz, 2H), 3.37 (t, J=6.3 Hz, 2H); ESIMS m/z (rel intensity) 353(MH⁺, 100). Anal. (C₂₃H₁₆N₂O₂.0.55H₂O) C, H, N.

cis-4-Carboxy-N-(3-chloropropyl)-3,4-dihydro-3-(4-methoxyphenyl)-1(2H)isoquinolone(8a). Homophthalic anhydride (6a) (3.065 g, 18.90 mmol) was added to achloroform (125 mL) solution of4-methoxybenzylidene-(3-chloro-1-propylamine) (7a) (4.000 g, 18.90 mmol)and the reaction mixture was allowed to stir at room temperature for 3h. The obtained precipitate was filtered, washed with chloroform (100mL), and dried to provide an off-white solid (4.723 g, 67%): mp 180-181°C. IR (KBr) 3437, 2957, 1740, 1622, 1598, 1573, 1514, 1479, 1258, and1173 cm⁻¹; ¹H NMR (CD₃OD) δ 8.10 (dd, J=7.6 Hz and 1.4 Hz, 1H), 7.63 (d,J=7.6 Hz, 1H), 7.55 (dt, J=7.4 Hz and 1.5 Hz, 1H), 7.50 (m, 1H), 6.97(m, 2H), 6.75 (m, 2H), 5.13 (d, J=6.3 Hz, 1H), 4.76 (d, J=6.2 Hz, 1H),3.98 (m, 1H), 3.70 (s, 3H), 3.61 (m, 2H), 3.22 (m, 1H), 2.13-2.01 (m,2H); ESIMS m/z (rel intensity) 374/376 (MH⁺, 100/33). Anal.(C₂₀H₂₀ClNO₄) C, H, N.

Benzylidene-(3-bromo-1-propylamine) (7b). The hydrobromide salt of3-bromopropylamine (5.364 g, 24.50 mmol) was treated with triethylamine(4 mL) in CHCl₃ (100 mL) and allowed to stir at room temperature for 5min. Benzaldehyde (2.000 g, 18.85 mmol) and magnesium sulfate (6.000 g)were added and the reaction mixture was allowed to stir at roomtemperature for 16 h. The reaction mixture was filtered and the filterpad was washed with CHCl₃ (50 mL). The filtrate was washed with water(3×50 mL), sat NaCl (50 mL), dried over sodium sulfate, and concentratedto provide a yellow oil (4.262 g, 100%). IR (film) 1645, 754, and 693cm⁻¹; ¹H NMR (CDCl₃) δ 8.34 (s, 1H), 7.76 (m, 2H), 7.44 (m, 3H), 3.78(dt, J=6.3 Hz and 1.3 Hz, 2H), 3.52 (t, J=6.5 Hz, 2H), 2.31 (pent, J=6.4Hz, 2H); ESIMS m/z (rel intensity) 226/228 (MH⁺, 100/91). Anal.(C₁₀H₁₂BrN) C, H, N.

cis-4-Carboxy-3,4-dihydro-N-(3-bromopropyl)-3-phenyl-7-nitro-1(2H)isoquinolone(8b). 4-Nitrohomophthalic anhydride (6b) (3.664 g, 17.69 mmol) was addedto a chloroform (125 mL) solution of benzylidene-(3-bromo-1-propylamine)(7b) (4.000 g, 17.69 mmol), and the reaction mixture was allowed to stirat room temperature for 1.25 h. The obtained precipitate was filtered,washed with chloroform (150 mL), and dried to provide a yellow solid(6.278 g, 82%): mp 158-160° C. IR (KBr) 3435, 3061, 1743, 1638, 1520,1349, and 1191 cm⁻¹; ¹H NMR (CD-30D) δ 8.90 (d, J=2.5 Hz, 1H), 8.38 (dd,J=8.7 Hz and 2.6 Hz, 1H), 7.98 (m, 1H), 7.25-7.19 (m, 3H), 7.07-7.03 (m,2H), 5.32 (d, J=6.2 Hz, 1H), 4.96 (d, J=6.2 Hz, 1H), 3.99 (m, 1H), 3.52(m, 2H), 3.26 (m, 1H), 2.26-2.12 (m, 2H); negative ion ESIMS m/z (relintensity) 431/433 [(M-H⁺)⁻, 12/9]. Anal. (C₁₉H₁₇BrN₂O⁵.1.0H₂O) C, H, N.

6-(3-Chloropropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(5bc). Thionyl chloride (2 mL) was added to a solution ofcis-4-carboxy-N-(3-chloropropyl)-3,4-dihydro-3-(4-methoxyphenyl)-1(2H)isoquinolone(8a) (0.510 g, 1.364 mmol) in benzene (40 mL). The reaction mixture washeated at reflux for 30 min, allowed to cool to room temperature, andconcentrated. The residue was diluted with nitrobenzene (20 mL), chilledin an ice bath, and aluminum chloride (0.364 g, 2.728 mmol) was added.The reaction mixture was removed from the bath and heated at 100° C. for1.5 h. Ice water (100 mL) was added and the solution was extracted withCHCl₃ (3×50 mL). The combined organic layer was washed with sat NaHCO₃(3×50 mL), sat NaCl (50 mL), and dried over sodium sulfate. The solutionwas concentrated, hexanes (250 mL) were added, and liquid was decanted.The obtained solid was washed with hexanes (100 mL) and the liquid wasagain decanted. The solid was purified by flash column chromatography(SiO₂), eluting with chloroform, to provide a purple-red solid (0.082 g,17%) that was precipitated from EtOAc/hexanes: mp 195-198° C. IR (KBr)1662, 1611, 1505, 1481, 1432, and 1299 cm⁻¹; ¹H NMR (CDCl₃) δ 8.67 (d,J=8.1 Hz, 1H), 8.31 (dd, J=8.2 Hz and 0.7 Hz, 1H), 7.73 (m, 1H), 7.66(d, J=8.4 Hz, 1H), 7.45 (m, 1H), 7.22 (d, J=2.6 Hz, 1H), 6.86 (dd, J=8.4Hz and 2.6 Hz, 1H), 4.67 (m, 2H), 3.89 (s, 3H), 3.83 (m, 2H), 2.43 (m,2H); CIMS m/z (rel intensity) 354/356 (MH⁺, 100/30). Anal. (C₂₀H₁₆ClNO₃)C, H, N.

6-(3-Bromopropyl)-5,6-dihydro-5,11-dioxo-3-nitro-11H-indeno[1,2-c]isoquinoline(5bb). Thionyl chloride (5 mL) was added to a solution ofcis-4-carboxy-3,4-dihydro-N-(3-bromopropyl)-3-phenyl-7-nitro-1(2H)isoquinolorie(8b) (1.000 g, 2.308 mmol) in benzene (50 mL). The reaction mixture washeated at reflux for 30 min, allowed to cool to room temperature, andconcentrated. The residue was diluted with nitrobenzene (30 mL), chilledin an ice bath, and aluminum chloride (0.616 g, 4.616 mmol) was added.The reaction mixture was removed from the bath and heated at 100° C. for1 h. Ice water (100 mL) was added and the solution was extracted withCHCl₃ (3×100 mL). The combined organic layer was washed with sat NaHCO₃(3×50 mL), sat NaCl (50 mL) and dried over sodium sulfate. The solutionwas concentrated, hexanes (900 mL) were added, and liquid was decanted.The obtained solid was washed with hexanes (100 mL) and the liquid wasagain decanted. The crude solid was purified by flash columnchromatography (SiO₂), eluting with chloroform, to provide an orangesolid (0.432 g, 45%): mp 258-260 (dec). IR (film) 1672, 1612, 1560,1503, 1428, and 1337 cm⁻¹; ¹H NMR (CDCl₃) δ 9.20 (d, J=2.4 Hz, 1H), 8.89(d, J=8.9 Hz, 1H), 8.52 (dd, J=9.0 Hz and 2.4 Hz, 1H), 7.92 (m, 1H),7.75 (m, 1H), 7.57-7.52 (m, 2H), 4.76 (m, 2H), 3.70 (t, J=6.2 Hz, 2H),2.54 (m, 2H); CIMS m/z (rel intensity) 413/415 (MH⁺, 100/82). Anal.(C₁₉H₁₃BrN₂O₄) C, H, N.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-ethyl}amine(12a). 2,2′-Diaminodiethylamine (11a) (0.3 g, 2.91 mmol) was added to astirred solution of indenobenzopyran 4d (2.17 g, 8.72 mmol) in CHCl₃(200 mL) and the mixture was stirred under reflux for 48 h. The reactionmixture was then cooled and the resultant orange solid was filteredthrough a sintered glass funnel and washed with chloroform (30 mL) toprovide pure bisindenoisoquinoline 12a (0.75 g, 46%) as an orange solid:mp 240-242° C. ¹H NMR (DMSO-d₆) δ 8.51 (d, J=8.9 Hz, 2H), 8.11 (d, J=7.7Hz, 2H), 7.76 (bs, 4H), 7.47 (bs, 4H), 7.36 (bs, 4H), 4.51 (bs, 4H),3.03 (bs, 4H); ESIMS m/z (rel intensity) (MH⁺, 100). Anal. Calcd forC₃₆H₂₅N₃O₄: C, 76.72; H, 4.47; N, 7.46. Found: C, 76.35; H, 4.45; N,7.39.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)}-(6-ethyl,6′-propyl)amine(12b). 2-Aminoethyl-3-aminopropylamine (11b) (0.2 g, 1.71 mmol) wasadded to a stirred solution of indenobenzopyran 4d (1.06 g, 4.27 mmol)in CHCl₃ (200 mL) and the reaction mixture was stirred under reflux for48 h. The reaction mixture was then cooled and the resultant orangesolid was filtered through a sintered glass funnel and washed withchloroform-methanol mixture (2:8, 50 mL) to provide purebisindenoisoquinoline 12b (0.72 g, 73%) as an orange solid: mp 250-252°C. ¹H NMR (CDCl₃) δ 8.69 (d, J=8.5 Hz, 2H), 8.29 (t, J=7.4 Hz, 2H), 7.70(t, J=7.4 Hz, 2H), 7.62 (m, 2H), 7.46-7.37 (m, 8H), 4.69 (t, J=7.3 Hz,2H), 4.61 (t, J=7.5 Hz, 2H), 3.16 (t, J=7.3 Hz, 2H), 2.89 (t, J=6.0 Hz,2H), 2.05 (m, 2H); ESIMS m/z (rel intensity) 578 (MH⁺, 100); HRESIMScalcd for (C₃₇H₂₇N₃O₄)H⁺: 578.2079. Found: 578.2087.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propyl}amine(12c). 3,3′-Diaminodipropylamine (11c) (0.3 g, 2.29 mmol) was added to astirred solution of indenobenzopyran 4d (1.7 g, 6.86 mmol) in CHCl₃ (200mL) and the mixture was stirred under reflux for 48 h. The bright orangereaction mixture was purified by flash column chromatography (SiO₂/CHCl₃to 3% MeOH in CHCl₃) to afford pure bisindenoisoquinoline 12c (0.54 g)in 40% yield as a dark orange solid: mp 223-225° C. ¹H NMR (CDCl₃) δ8.65 (d, J=8.1 Hz, 2H), 8.27 (d, J=8.1 Hz, 2H), 7.67 (t, J=7.1 Hz, 4H),7.57 (d, J=7.0 Hz, 2H), 7.41 (t, J=7.1 Hz, 4H), 7.33 (t, J=7.1 Hz, 2H),4.62 (t, J=7.3 Hz, 4H), 2.84 (t, J=6.4 Hz, 4H), 2.08 (m, 4H); ¹H NMR(DMSO-d₆) δ 8.53 (d, J=7.8 Hz, 2H), 8.18 (d, J=7.9 Hz, 2H), 7.87 (d,J=7.4 Hz, 2H), 7.79 (t, J=7.6 Hz, 2H), 7.54 (t, J=5.8 Hz, 4H), 7.46 (m,4H), 4.53 (t, J=6.9 Hz, 4H), 2.80 (bs, 4H), 1.99 (m, 4H); ESIMS m/z (relintensity) 592 (MH⁺, 100). Anal. Calcd for C₃₈H₂₉N₃O₄.1.6H₂O: C, 73.56;H, 5.23; N, 6.77. Found: C, 73.18; H, 4.93; N, 6.47.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propyl}methylamine(12d). 3,3′-Diamino-N-methyl dipropylamine (11d) (0.10 g, 0.69 mmol) wasadded to a stirred solution of indenobenzopyran 4d (0.38 g, 1.52 mmol)in CHCl₃ (150 mL) and the reaction mixture was stirred under reflux for48 h. The reaction mixture was cooled to room temperature and purifiedby flash column chromatography (SiO₂/CHCl₃ to 5% MeOH in CHCl₃) toprovide bisindenoisoquinoline 12d (340 mg, 82%) as a red solid: mp230-232° C. ¹H NMR (CDCl₃) δ 8.67 (d, J=8.1 Hz, 2H), 8.30 (d, J=7.6 Hz,2H), 7.72-7.66 (dt, J=8.3 and 2.8 Hz, 4H), 7.59 (d, J=7.1 Hz, 2H),7.48-7.40 (q, J=7.5 Hz, 4H), 7.33 (t, 7.3 Hz, 2H), 4.63 (t, J=8.0 Hz,4H), 2.66 (t, J=6.5 Hz, 4H), 2.37 (s, 3H), 2.13-2.04 (m, 4H); ESIMS m/z(rel intensity) 606 (MH⁺, 100). Anal. Calcd for C₃₉H₃₁N₃O₄.0.4 CHCl₃: C,72.42; H, 4.84; N, 6.43. Found: C, 72.67; H, 5.05; N, 6.32.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)}-(6-propyl,6′-butyl)amine(12e). 4-Aminobutyl-3-aminopropylamine (11e) (0.2 g, 1.38 mmol) wasadded to a stirred solution of indenobenzopyran 4d (0.75 g, 3.03 mmol)in CHCl₃ (200 mL) and the reaction mixture was stirred under reflux for48 h. The reaction mixture was then cooled and the resultant orangesolid was filtered through a sintered glass funnel and washed withchloroform-methanol mixture (5:1, 50 mL) to provide purebisindenoisoquinoline 12e (0.63 g, 76%) as an orange solid: mp 228-230°C. ¹H NMR (CDCl₃) δ 8.67 (d, J=8.1 Hz, 2H), 8.28 (d, J=8.1 Hz, 2H),7.70-7.65 (m, 2H), 7.59 (d, J=6.8 Hz, 2H), 7.52 (d, J=7.4 Hz, 2H),7.45-7.24 (m, 6H), 4.61 (t, J=7.3 Hz, 2H), 4.55 (t, J=7.9 Hz, 2H), 2.84(t, J=6.5 Hz, 2H), 2.80 (t, J=6.8 Hz, 2H), 2.15-2.10 (m, 2H), 2.00-1.95(m, 2H), 1.84-1.77 (m, 2H); ESIMS m/z (rel intensity) 606 (MH⁺, 100);HRESIMS calcd for (C₃₉H₃₁N₃O₄)H⁺: 606.2393. Found: 606.2402.

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-(6-ethyl-tert-BOCamino)}propane(13a). N,N′-Bis(2-aminoethyl)-1,3-propanediamine (11g) (0.10 g, 0.62mmol) was added to a stirred solution of indenobenzopyran 4d (0.34 g,1.37 mmol) in CHCl₃ (150 mL) and the reaction mixture was stirred underreflux for 72 h, providing bisindenoisoquinoline 12g as a crudeintermediate. After allowing the reaction mixture to cool to roomtemperature, Et₃N (0.35 mL, 2.50 mmol) and Boc₂O (0.34 g, 1.56 mmol)were added, and the reaction mixture was stirred at room temperature for8 h. The crude reaction mixture was purified by flash columnchromatography (SiO₂/20% EtOAc in hexane, then 1-5% MeOH in CHCl₃) toprovide Doc-protected bisindenoisoquinoline 13a (380 mg, 74%) as anorange solid: mp 238-240° C. ¹H NMR (CDCl₃) δ 8.63 (d, J=8.0 Hz, 1H),8.16 (d, J=7.4 Hz, 2H), 7.65-7.58 (m, 5H), 7.53-7.45 (m, 2H), 7.38-7.29(m, 5H), 4.63 (bs, 4H), 3.62 (bs, 4H), 3.32 (bs, 4H), 1.90 (bs, 2H),1.41 (s, 18H); ESIMS m/z (rel intensity) 821 (MH⁺, 10), 721 (MH⁺-Boc,100). Anal. Calcd for C₄₉H₄₈N₄O₈: C, 71.69; H, 5.89; N, 6.82. Found: C,71.35; H, 5.99; N, 6.68.

Bis-1,2-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-(6-propyl-tert-BOCamino)}ethane(13b). N,N′-Bis(3-aminopropyl)-1,2-ethanedimaine (11h) (0.16 g, 0.85mmol) was added to a stirred solution of indenobenzopyran 4d (0.46 g,1.87 mmol) in CHCl₃ (150 mL) and the reaction mixture was stirred underreflux for 72 h, providing bisindenoisoquinoline 12h as a crudeintermediate. Upon allowing the reaction mixture to cool to roomtemperature, Et₃N (0.6 mL, 4.24 mmol) and Boc₂O (0.56 g, 2.60 mmol) wereadded to the reaction mixture and the mixture was allowed to stir atroom temperature for 8 h. The crude reaction mixture was purified byflash column chromatography (SiO₂/20% EtOAc in hexane, then 1-5% MeOH inCHCl₃) to provide Boc-protected bisindenoisoquinoline 13b (550 mg, 76%)as an orange solid: mp 106-108° C. ¹H NMR (CDCl₃) δ 8.60 (bs, 2H), 8.23(bs, 2H), 7.65 (bs, 2H), 7.55 (d, J=6.7 Hz, 2H), 7.40-7.32 (m, 8H), 4.48(bs, 4H), 3.45 (bs, 8H), 2.12 (bs, 4H), 1.44 (s, 9H), 1.39 (s, 9H);ESIMS m/z (rel intensity) 835 (MH⁺, 22), 735 (MH⁺-Boc, 100). Anal. Calcdfor C₅₀H₅₀N₄O₈.0.3H₂O: C, 71.46; H, 6.07; N, 6.67. Found: C, 71.15; H,6.19; N, 6.61.

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-(6-propyl-tert-BOCamino)}propane(13c). N,N′-Bis(3-aminopropyl)-1,3-propanediamine (11j) (0.15 g, 0.74mmol) was added to a stirred solution of indenobenzopyran 4d (0.40 g,1.63 mmol) in CHCl₃ (150 mL) and the reaction mixture was stirred underreflux for 72 h, providing bisindenoisoquinoline 12j as a crudeintermediate. Upon allowing the reaction mixture to cool to roomtemperature, Et₃N (0.53 mL, 3.78 mmol) and Boc₂O (0.49 g, 2.27 mmol)were added to the reaction mixture and the mixture was allowed to stirat room temperature for 8 h. The crude reaction mixture was purified byflash column chromatography (SiO₂/20% EtOAc in hexane, then 1-5% MeOH inCHCl₃) to provide Boc-protected bisindenoisoquinoline 13c (450 mg, 70%)as an orange solid: mp 86-88° C. ¹H NMR (CDCl₃) δ 8.63 (d, J=8.1 Hz,2H), 8.24 (d, J=7.6 Hz, 2H), 7.65 (t, J=7.3 Hz, 2H), 7.55 (d, J=6.7 Hz,2H), 7.40-7.31 (m, 8H), 4.49 (bs, 4H), 3.44 (bs, 4H), 3.27 (apparent t,J=6.2 Hz, 4H), 2.08 (bs, 4H), 1.86 (bs, 2H), 1.41 (bs, 18H); ESIMS m/z(relative intensity) 849 (MH⁺, 3), 749 (MH⁺−Boc, 37), 649 (MH⁺−2xBoc,100). Anal. Calcd for C₅₁H₅₂N₄O₈.0.5H₂O: C, 71.39; H, 6.23; N, 6.53.Found: C, 70.99; H, 620; N, 6.62.

Bis-1,4-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-e]isoquinoline)-(6-propyl-tent-BOCamino)}butane(13d). N,N′-Bis(3-aminopropyl)-1,4-butanediamine (11k) (0.10 g, 0.50mmol) was added to a stirred solution of indenobenzopyran 4d (0.27 g,1.09 mmol) in CHCl₃ (150 mL) and the reaction mixture was stirred underreflux for 72 h, providing bisindenoisoquinoline 12k as a crudeintermediate. Upon allowing the reaction mixture to cool to roomtemperature, Et₃N (0.28 mL, 2.00 mmol) and Boc₂O (0.27 g, 1.25 mmol)were added to the reaction mixture and the mixture was allowed to stirat room temperature for 8 h. The crude reaction mixture was purified byflash column chromatography (SiO₂/20% EtOAc in hexane, and then 1-5%MeOH in CHCl₃) to provide Boc-protected bisindenoisoquinoline 13d (350mg, 82%) as an orange solid: mp 92-94° C. ¹H NMR (CDCl₃) δ 8.66 (d,J=8.1 Hz, 2H), 8.28 (d, J=8.0 Hz, 2H), 7.68 (t, J=7.7 Hz, 2H), 7.60 (d,J=7.1 Hz, 2H), 7.43-7.34 (m, 8H), 4.51 (t, J=8.3 Hz, 4H), 3.44 (bs, 4H),3.28 (bs, 4H), 2.11 (m, 4H), 1.50 (bs, 4H), 1.41 (s, 18H); ESIMS m/z(rel intensity) 863 (MH⁺, 13), 763 (MH⁺−Boc, 100). Anal. Calcd forC₅₂H₅₄N₄O₈.0.9 H₂O: C, 71.04; H, 6.40; N, 6.37. Found: C, 70.77; H,6.39; N, 6.26.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)}-(6-ethyl,6′-propyl)ammoniumTrifluoroacetate (14a). Bisindenoisoquinoline 12b (0.5 g, 0.87 mmol) wasdissolved in neat CF₃COOH (30 mL) and the reaction mixture was stirredat room temperature for 1 h. The reaction mixture was concentrated,diluted with chloroform (50 mL), and the resultant solid was filteredthrough a sintered glass funnel and further washed with methanol (50 mL)to give bisindenoisoquinoline 14a (0.48 g, 80%) as a red solid: mp240-242° C. ¹H NMR (DMSO-d₆) δ 8.71 (bs, 1H, —NH—), 8.56 (d, J=7.8 Hz,2H), 8.17 (d, J=8.6 Hz, 2H), 7.83-7.75 (m, 4H), 7.57-7.50 (m, 8H), 4.79(bs, 2H), 4.57 (bs, 2H), 3.46 (bs, 2H), 3.17 (bs, 2H), 2.18 (bs, 2H);ESIMS m/z (rel intensity) 578 (MH⁺—CF₃COOH, 100). Anal. Calcd forC₃₉H₂₈N₃O₆F₃.0.3 H₂O: C, 67.20; H, 4.14; N, 6.03. Found: C, 66.85; H,4.12; N, 5.93.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)}-(6-propyl,6′-butyl)amineHydrochloride (14b). 2 M HCl in ether (6.2 in L, 2.4 mmol) was added toa stirred solution of bisindenoisoquinoline 12e (0.5 g, 0.83 mmol) inchloroform (100 mL) and the reaction mixture was stirred at roomtemperature for 2 h. The reaction mixture was filtered through asintered glass funnel and the solid was washed with chloroform (50 mL)and methanol (50 mL) to give bisindenoisoquinoline hydrochloride 14b(0.44 g, 83%) as an orange solid: mp 280-282° C. (dec). ¹H NMR (DMSO-d₆)δ 8.66 (bs, 1H), 8.54 (d, J=7.9 Hz, 2H), 8.18 (d, J=8.6 Hz, 2H),7.83-7.7 d 0 (m, 4H), 7.58-7.40 (m, 8H), 4.54-4.42 (m, 4H), 3.06 (bs,2H), 2.96 (bs, 2H), 2.16 (bs, 2H), 1.84 (bs, 2 II), 1.76 (bs, 2H); ESIMSm/z (rel intensity) 606 (MH⁺, 100). Anal. Calcd for C₃₉H₃₂N₃O₄Cl.1.1H₂O: C, 70.76; H, 5.21; N, 6.35. Found: C, 70.48; H, 5.12; N, 6.23.

Bis-1,2-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-ethylamino}ethaneBis(trifluoroacetate) (14c). N,N′-Bis(2-aminoethyl)-1,2-ethanediamine(11f) (0.4 g, 2.74 mmol) was added to a stirred solution ofindenobenzopyran 4d (1.49 g, 6.02 mmol) in CHCl₃ (200 mL) and thereaction mixture was stirred under reflux for 48 h. The reaction mixturewas then cooled and the resultant orange solid was filtered through asintered glass funnel and washed with chloroform (50 mL) to providebisindenoisoquinoline 12f (0.57 g, 69%) as an insoluble orange solid.Intermediate 12f (0.5 g, 0.83 mmol) was dissolved in neat CF₃COOH (30mL) and stirred at room temperature for 30 min. The reaction mixture wasconcentrated, diluted with chloroform (50 mL), and filtered through asintered glass funnel to provide bisindenoisoquinoline 14c (0.57 g, 83%)as an orange solid: mp 230-232° C. ¹H NMR (DMSO-d₆) δ 8.97 (bs, 2H),8.59 (d, J=8.1 Hz, 2H), 8.23 (d, J=8.0 Hz, 2H), 7.89-7.83 (td, J=1.2 and8.3 Hz, 2H), 7.76 (d, J=6.8 Hz, 2H), 7.63-7.50 (m, 8H), 4.83 (bs, 4H),3.52 (bs, 4H), 3.32 (bs, 4H); ESIMS m/z (rel intensity) 607 (MH⁺, 100).Anal. Calcd for C₄₂H₃₂N₄O₈F₆.0.4H₂O: C, 59.92; H, 3.93; N, 6.66. Found:C, 59.56; H, 4.04; N, 6.62.

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-ethylamino}propaneBis(trifluoroacetate) (14d). Boc-protected bisindenoisoquinoline 13a(0.3 g, 0.36 mmol) was dissolved in neat CF₃COOH (30 mL) and the mixturewas stirred at room temperature for 1 h. The reaction mixture wasconcentrated and the resultant solid was diluted with chloroform (50 mL)and filtered through a sintered glass funnel to providebisindenoisoquinoline 14d (0.28 g, 92%) as an orange solid: mp 244-246°C. ¹H NMR (DMSO-d₆) δ 8.93 (bs, 2H), 8.60 (d, J=8.4 Hz, 2H), 8.23 (d,J=7.9 Hz, 2H), 7.87 (t, J=7.5 Hz, 2H), 7.79 (d, J=7.8 Hz, 2H), 7.60-7.52(m, 8H), 4.82 (bs, 4H), 3.47 (bs, 4H), 3.07 (bs, 4H), 1.95 (bs, 2H);ESIMS m/z (rel intensity) 621 (MH, 100), 274 (7). Anal. Calcd forC₄₃H₃₄F₆N₄O₈.1.7 H₂O: C, 58.73; H, 4.29; N, 6.37. Found: C, 58.38; H,4.32; N, 6.26.

Bis-1,2-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}ethaneBis(trifluoroacetate) (14e). Boc-protected bisindenoisoquinoline 13b(0.5 g, 0.79 mmol) was dissolved in neat CF₃COOH (30 mL) and the mixturewas stirred at room temperature for 30 min. The reaction mixture wasconcentrated, diluted with chloroform (50 mL), and the resultant solidwas filtered through a sintered glass funnel to affordbisindenoisoquinoline 14e (0.61 g, 90%) as a pale red solid: mp 220-222°C. ¹H NMR (DMSO-d₆) δ 8.84 (bs, 2H), 8.57 (d, J=8.1 Hz, 2H), 8.20 (d,J=7.8 Hz, 2H), 7.85-7.77 (m, 4H), 7.60-7.48 (m, 8H), 4.57 (t, J=6.6 Hz,4H), 3.23 (bs, 4H), 3.18 (bs, 4H), 2.16 (m, 4H); ESIMS m/z (relintensity) 635 (MH⁺, 61). Anal. Calcd for C₄₄H₃₆N₄O₈F₆.1.4H₂O: C, 59.51;H, 4.40; N, 6.31. Found: C, 59.15; H, 4.06; N, 6.06.

Bis-1,4-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propyl}piperazineBis(trifluoroacetate) (14f). 1,4-Bis(3-aminopropyl)piperazine 11i (0.10g, 0.50 mmol) was added to a stirred solution indenobenzopyran 4d (0.27g, 1.10 mmol) in CHCl₃ (150 mL) and the reaction mixture was stirredunder reflux for 60 h. The reaction mixture was then cooled and theresultant red solid was filtered off through a sintered glass funnel,washed with chloroform (50 mL) and dried to provide intermediate 12i.This compound was further treated with CF₃COOH (40 mL) and the mixturewas stirred at room temperature for 2 h. The reaction mixture wasconcentrated, diluted with chloroform (50 mL), and the resultant solidwas filtered and washed with methanol-chloroform (1:9) to providebisindenoisoquinoline 14f (430 mg, 86%) as red solid: mp 256-258° C. ¹HNMR (CDCl₃) δ 8.57 (d, J=8.0 Hz, 2H), 8.21 (d, J=8.1 Hz, 2H), 7.85-7.77(m, 4H), 7.58-7.48 (m, 8H), 4.55 (bs, 4H), 3.34 (bs, 4H), 3.02 (bs, 4H),2.72 (bs, 2H), 2.47 (bs, 2H, merged with DMSO-d₆ protons), 2.09 (bs,4H); ESIMS m/z (rel intensity) 661 (MH⁺, 100). Anal. Calcd forC₄₆H₃₈F₆N₄O₈.0.4 H₂O: C, 61.66; H, 4.37; N, 6.25. Found: C, 61.27; H,4.61; N, 6.18.

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}propaneBis(trifluoroacetate) (14g). Boc-protected bisindenoisoquinoline 13c(0.3 g, 0.35 mmol) was dissolved in neat CF₃COOH (30 mL) and the mixturewas stirred at room temperature for 1 h. The reaction mixture wasconcentrated and the resultant solid was diluted with chloroform (50 mL)and filtered through a sintered glass funnel to providebisindenoisoquinoline 14g (0.27 g, 89%) as an orange solid: mp 225-227°C. ¹H NMR (DMSO-d₆) δ 8.66 (bs, 2H), 8.56 (d, J=8.1 Hz, 2H), 8.19 (d,J=7.9 Hz, 2H), 7.79 (t, J=7.9 Hz, 4H), 7.59-7.48 (m, 8H), 4.57 (bs, 4H),3.09 (bs, 4H), 2.96 (bs, 4H), 2.15 (bs, 4H), 1.87 (bs, 2H); ESIMS m/z(relative intensity) 649 (MH⁺, 100). Anal. Calcd for C₄₅H₃₈F₆N₄O₈.1.3H₂O: C, 60.04; H, 4.55; N, 6.22. Found: C, 59.71; H, 4.41; N, 6.03.

Bis-1,4-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}butaneBis(trifluoroacetate) (14h). Boc-protected bisindenoisoquinoline 13d(0.3 g, 0.35 mmol) was dissolved in neat CF₃COOH (20 mL) and the mixturewas stirred at room temperature for 1 h. The reaction mixture wasconcentrated and the resultant solid was diluted with chloroform (50 mL)and filtered through a sintered glass funnel to providebisindenoisoquinoline 14h (0.28 g, 90%) as an orange solid: mp 236-238°C. ¹H NMR (DMSO-d₆) δ 8.58 (d, J=8.0 Hz, 2H), 8.52 (bs, 2H), 8.20 (d,J=8.1 Hz, 2H), 7.84-7.78 (m, 4H), 7.60-7.49 (m, 8H), 4.57 (t, J=6.6 Hz,4H), 3.08 (bs, 4H), 2.92 (bs, 4H), 2.16 (m, 4H), 1.59 (bs, 4H); ESIMSm/z (rel intensity) 663 (MH⁺, 100). Anal. Calcd for C₄₆H₄₀F₆N₄O₈.0.4H₂O: C, 61.52; H, 4.58; N, 6.24. Found: C, 61.22; H, 4.62; N, 6.09.

Bis{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-ethylamino-ethyl}amineTris(trifluoroacetate) (14i).N-(2-aminoethyl)-N′-[(2-aminoethyl)aminoethyl)]-1,2-ethanediamine (11l)(0.20 g, 1.06 mmol) was added to a stirred solution of indenobenzopyran4d (0.58 g, 2.32 mmol) in CHCl₃ (150 mL) and the reaction mixture wasstirred under reflux for 4 days, providing bisindenoisoquinoline 121 asa crude intermediate. Upon allowing the reaction mixture to cool to roomtemperature, Et₃N (0.86 mL, 6.13 mmol) and Boc₂O (0.89 g, 4.09 mmol)were added and the mixture was allowed to stir at room temperature for12 h. The crude reaction mixture was purified by flash columnchromatography (SiO₂/20% EtOAc in hexane, then 1-3% MeOH in CHCl₃) toprovide Boc-protected bisindenoisoquinoline 13e (0.61 g, 61%), which wasfurther treated with neat CF₃COOH (30 mL) and stirred at roomtemperature for 3 h. The reaction mixture was concentrated and theresultant solid was diluted with chloroform (50 mL) and filtered througha sintered glass funnel to provide bisindenoisoquinoline 14i (0.42 g,66%) as red solid: mp 198-200° C. (dec). ¹H NMR (DMSO-d₆) δ 8.55 (d,J=8.1 Hz, 2H), 8.20 (d, J=7.6 Hz, 2H), 7.85-7.76 (m, 4H), 7.59-7.48 (m,8H), 4.81 (bs, 4H), 3.54 (bs, 4H), 3.32 (bs, 8H); ESIMS m/z (relintensity) 650 (MH⁺, 100). Anal. Calcd for C₄₆H₃₈N₅O₁₀F₉.0.6 CH₂Cl₂NH₃:C, 53.15; H, 3.93; N, 7.45. Found: C, 53.16; H, 4.27; N, 7.81.

Bis-1,2-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-ethylamino-ethylamino}ethaneTetra(trifluoroacetate) (14j).N,N′-Bis{(2-aminoethyl)aminoethyl)}-1,2-ethanediamine (11m) (0.20 g,0.86 mmol) was added to a stirred solution of indenobenzopyran 4d (0.47g, 1.89 mmol) in CHCl₃ (150 mL) and the reaction mixture was stirredunder reflux for 4 days, providing bisindenoisoquinoline 12m as a crudeintermediate. Upon allowing the reaction mixture to cool to roomtemperature, Et₃N (1.21 mL, 8.67 mmol) and Boc₂O (0.95 g, 4.34 mmol)were added to the reaction mixture and the mixture was allowed to stirat room temperature for 12 h. The crude reaction mixture was purified byflash column chromatography (SiO₂/20% EtOAc in hexane, then 1-3% MeOH inCHCl₃) to provide Boc-protected bisindenoisoquinoline 13f (0.62 g, 66%),which was further treated with neat CF₃COOH (30 mL) and stirred at roomtemperature for 3 h. The reaction mixture was concentrated and theresultant solid was diluted with chloroform (50 mL) and filtered througha sintered glass funnel to provide bisindenoisoquinoline 14j (0.48 g,49%) as red solid: mp 206-208° C. (dec). ¹H NMR (DMSO-d₆) δ 8.55 (t,J=8.4 Hz, 2H), 8.19 (t, J=8.0 Hz, 2H), 7.82-7.61 (m, 4H), 7.59-7.51 (m,8H), 4.81. (bs, 4H), 3.52 (bs, 4H), 3.27 (bs, 4H), 3.19-3.13 (bs, 8H);ESIMS m/z (rel intensity) 693 (MH⁺, 100). Anal. Calcd forC₅₀H₄₄N₆O₁₂F₁₂.0.6H₂O: C, 51.78; H, 3.93; N, 7.25. Found: C, 51.41; H,4.17; N, 7.53.

Bis-1,3-{(5,6-dihydro-5,11-diketo-2,3-dimethoxy-11H-indeno[1,2-c]isoquinoline)-6-ethylamino}propaneBis{trifluoroacetate) (14k). N,N′-Bis(2-aminoethyl)-1,3-propanediamine(11g) (0.050 g, 0.309 mmol) was added to a solution of2,3-dimethoxybenz[d]indeno[1,2-b]pyran-5,11-dione (4a) (0.200 g, 0.649mmol) in CHCl₃ (50 mL). The solution was heated at reflux for 72 h andcooled to room temperature. Triethylamine (0.17 mL) and Boc₂O (0.270 g,1.236 mmol) were added to the solution and stirring was continued atroom temperature for 16 h. The solution was washed with water (2×25 mL)and sat NaCl (25 mL), dried over sodium sulfate, and concentrated. Thecrude red solid was purified by flash column chromatography (SiO₂/CHCl₃to 3% MeOH in CHCl₃) followed by precipitation from CH₂Cl₂-hexanes toprovide a pink solid. The obtained pink solid was diluted withtrifluoroacetic acid (30 mL) and the mixture was stirred at roomtemperature for 16 h. The solution was concentrated, diluted with CHCl₃(50 mL) and filtered to provide a red solid (0.257 g, 86%): mp 225-228°C. IR (KBr) 3437, 1652, 1553, 1513, 1429, 1268, 1204, and 1021 cm⁻¹; ¹HNMR (DMSO-d₆) δ 7.96 (s, 2H), 7.72-7.69 (bs, 2H), 7.52-7.43 (m, 8H),4.71 (bs, 4H), 3.92 (s, 6H), 3.81 (s, 6H), 3.06 (bs, 4H), 1.99 (bs, 2H);ESIMS m/z (rel intensity) 741 (MH⁺, 100). Anal. Calcd forC₄₇H₄₂F₆N₄O₁₂.4 H₂O: C, 54.23; H, 54.83; N, 5.38. Found: C, 54.63; H,4.49; N, 5.47.

Bis-1,3-{(5,6-dihydro-5,11-diketo-2,3-dimethoxy-11H-indeno[1,2-c]isoquinoline)-6-propylamino}propaneBis{trifluoroacetate) (141). N,N′-Bis(3-aminopropyl)-1,3-propanediamine(11j) (0.058 g, 0.309 mmol) was added to a solution of indenobenzopyran4a (0.200 g, 0.649 mmol) in CHCl₃ (50 mL). The solution was heated atreflux for 72 h and cooled to room temperature. Triethylamine (0.17 mL)and Boc₂O (0.270 g, 1.236 mmol) were added to the solution and stirringwas continued at room temperature for 16 h. The solution was washed withwater (2×25 mL) and sat NaCl (25 mL), dried over sodium sulfate, andconcentrated. The crude orange solid was purified by flash columnchromatography (SiO₂/CHCl₃ to 3% MeOH in CHCl₃) followed byprecipitation from EtOAc to provide an orange solid. The obtained orangesolid was diluted with trifluoroacetic acid (30 mL) and the mixture wasstirred at room temperature for 16 h. The solution was concentrated,diluted with CHCl₃ (50 mL) and filtered to provide a red solid (0.221 g,72%): mp 273-276° C. (dec). IR (KBr) 3436, 1639, 1553, 1512, 1478, 1429,1267, 1184, and 1022 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.43 (bs, 4H), 8.00 (s,2H), 7.76 (d, J=7.58 Hz, 2H), 7.59-7.45 (m, 8H), 4.56 (bs, 4H), 3.93 (s,6H), 3.85 (s, 6H), 3.09 (bs, 4H), 2.98 (bs, 4H), 2.15 (bs, 4H), 1.86(bs, 2H); ESIMS m/z (rel intensity) 769 (MH⁺, 100). Anal. Calcd forC₄₉H₄₆F₆N₄O₁₂.6 H₂O: C, 53.26; H, 5.29; N, 5.07. Found: C, 52.88; H,4.96; N, 5.21.

Bis-1,3-{(5,6-dihydro-5,11-diketo-3-nitro-11H-indeno[1,2-c]isoquinoline)-6-ethylamino}propaneBis{trifluoroacetate) (14m). N,N′-Bis(2-aminoethyl)-1,3-propanediamine(11g) (0.056 g, 0.349 mmol) was added to a solution of indenobenzopyran4c (0.225 g, 0.767 mmol) in CHCl₃ (50 mL). The solution was heated atreflux for 72 h and cooled to room temperature. Triethylamine (0.19 mL)and Boc₂O (0.305 g, 1.396 mmol) were added to the solution and stirringwas continued at room temperature for 16 h. The solution was washed withwater (2×30 mL) and sat NaCl (30 mL), dried over sodium sulfate, andconcentrated. The crude orange solid was purified by flash columnchromatography (SiO₂/CHCl₃ to 3% MeOH in CHCl₃) to provide an orangesolid. The orange solid was diluted with trifluoroacetic acid (40 mL)and the mixture was stirred at room temperature for 24 h. The solutionwas concentrated, diluted with CHCl₃ (50 mL) and filtered to provide anorange solid (0.221 g, 67%): mp 227-230° C. (dec). IR (KBr) 3433, 3087,3022, 2819, 1679, 1615, 1560, 1505, 1429, 1138, and 1200 cm⁻¹; ¹H NMR(CDCl₃) δ 8.90 (bs, 4H), 8.79 (d, J=9.14 Hz, 2H), 8.66 (d, J=9.07 Hz,2H), 7.93 (d, J=6.54 Hz, 2H), 7.74 (d, J=7.17 Hz, 2H), 7.67 (m, 4H),4.87 (bs, 4H), 3.49 (bs, 4H), 3.09 (bs, 4H), 1.91 (bs, 2H); ESIMS m/z(rel intensity) 711 (MH⁺, 100). Anal. Calcd for C₄₃H₃₂F₆N₆O₁₂.0.5H₂O: C,54.49; H, 3.51; N, 8.87. Found: C, 54.24; H, 3.80; N, 8.86.

Bis-1,3-{(5,6-dihydro-5,11-diketo-3-nitro-11H-indeno[1,2-c]isoquinoline)-6-propylamino}propaneBis{trifluoroacetate) (14n). N,N′-Bis(3-aminopropyl)-1,3-propanediamine(11j) (0.064 g, 0.341 mmol) was added to a solution of indenobenzopyran4c (0.200 g, 0.682 mmol) in CHCl₃ (75 mL). The solution was heated atreflux for 72 h and cooled to room temperature. Triethylamine (0.19 mL)and Boc₂O (0.298 g, 1.364 mmol) were added to the solution and stirringwas continued at room temperature for 16 h. The solution was washed withwater (2×30 mL) and sat NaCl (30 mL), dried over sodium sulfate, andconcentrated. The crude orange solid was purified by flash columnchromatography (SiO₂/CHCl₃ to 3% MeOH in CHCl₃) to provide an orangesolid. The obtained orange solid was diluted with trifluoroacetic acid(40 mL) and stirred at room temperature for 2 h. The solution wasconcentrated, diluted with CHCl₃ (50 mL) and filtered to provide anorange solid (0.206 g, 62%): mp 220-223° C. IR (KBr) 1678, 1614, 1505,1339, 1203, and 1132 cm⁻¹; ¹H NMR (CDCl₃) δ 8.88 (d, J=2.5 Hz, 2H), 8.75(d, J=9.0 Hz, 2H), 8.63 (bs, 2H), 8.60 (dd, J=9.0 Hz and 2.5 Hz, 2H),7.92 (d, J=6.5 Hz, 2H), 7.70-7.61 (m, 6H), 4.64 (t, J=5.9 Hz, 4H), 3.15(bs, 4H), 2.98 (bs, 4H), 2.19 (bs, 2H); ESIMS m/z (rel intensity) 739(MH⁺, 100). Anal. Calcd for C₄₅H₃₆F₆N₆O₁₂.3 H₂O: C, 52.95; H, 4.15; N,8.23. Found: C, 53.33; H, 4.32; N, 8.60.

1,3-{6-(3-tert-Butyloxycarbonylamino-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline}-{5′,6′-dihydro-6′-[(3′-tert-butyloxycarbonylamino)-1′-propyl]-5′,11′-dioxo-11′H-indeno[1,2-c]isoquinoline}propane(16a). 5 M NaOH (aq) was added slowly to a solution ofindenoisoquinoline hydrochloride A (1.0 g, 1.58 mmol) in awater-chloroform solution (2:1, 250 mL), which was prepared according toNagarajan, M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;Cushman, M., Design, Synthesis, and Biological Evaluation ofIndenoisoquinoline Topoisomerase I Inhibitors Featuring Polyamine SideChains on the Lactam Nitrogen. J. Med. Chem. 2003, 46, 5712-5724, thedisclosure of which is hereby incorporated by reference. At a pH of 7-8,the organic layer was separated and the aqueous layer was extracted withchloroform (3×100 mL). The combined organic layers were washed withwater (100 mL), sat NaCl (100 mL), dried over Na₂SO₄, and concentrated.Indenobenzopyran 4d (0.43 g, 1.74 mmol) was added to a solution of thecrude indenoisoquinoline triamine A (0.70 g, 1.34 mmol) in chloroform(200 mL) and the reaction mixture was heated at reflux for 4 days. Thereaction mixture containing crude, unsymmetrical bisindenoisoquinoline15a was cooled to room temperature, Et₃N (0.93 mL, 6.64 mmol) and Boc₂O(0.87 g, 3.98 mmol) were added, and the solution was allowed to stir atroom temperature for 12 h. The crude reaction mixture was purified byflash column chromatography (SiO₂/20% EtOAc in hexane, then 1-3% MeOH inCHCl₃) to provide Boc-protected bisindenoisoquinoline 16a (0.72 g, 48%)as purple solid: mp 120-122° C. ¹H NMR (CDCl₃) δ 8.64 (d, J=8.3 Hz, 1H),8.24 (d, J=5.7 Hz, 1H), 7.94 (s, 1H), 7.67 (t, J=7.4 Hz, 1H), 7.58 (s,1H), 7.56 (s, 1H), 7.42-7.34 (m, 5H), 6.99 (s, 1H), 6.05 (s, 2H), 4.49(bs, 2H), 4.40 (bs, 2H), 4.01 (s, 3H), 3.92 (s, 3H), 3.45 (bs, 4H), 3.29(bs, 4H), 2.10 (bs, 4H), 1.87 (m, 2H), 1.42 (s, 18H); ESIMS m/z (relintensity) 953 (MH⁺, 30), 853 (MH⁺-Boc, 100). Anal. Calcd forC₅₄H₅₆N₄O₁₂.0.9 CHCl₃: C, 62.18; H, 5.41; N, 5.28. Found; C, 62.08; H,5.36; N, 5.15.

1,3-{6-(3-Amino-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline}-{5′,6′-Dihydro-6′-(3′-amino-1′-propyl)-5′,11′-dioxo-11′H-indeno[1,2-c]isoquinoline}propaneBis(trifluoroacetate) (17a). Boc-protected bisindenoisoquinoline 16a(0.55 g, 0.58 mmol) was dissolved in neat CF₃COOH (30 mL) and themixture was stirred at room temperature for 3 h. The reaction mixturewas concentrated and the resultant solid was diluted with chloroform (50mL) and filtered through a sintered glass funnel to providebisindenoisoquinoline 17a (0.43 g, 76%) as purple solid: mp 218-220° C.¹H NMR (DMSO-d₆) δ 8.65 (bs, 2H), 8.53 (d, J=8.0 Hz, 1H), 8.17 (d, J=7.2Hz, 1H), 7.80-7.75 (m, 3H), 7.55-7.50 (m, 4H), 7.39 (s, 1H), 7.32 (s,1H), 7.02 (s, 1H), 6.18 (s, 2H), 4.55 (bs, 2H), 4.45 (bs, 2H), 3.86 (s,3H), 3.81 (s, 3H), 3.07-2.98 (bs, 8H), 2.14 (bs, 4H), 1.89 (bs, 2H);ESIMS m/z (rel intensity) 753 (MH⁺, 100). Anal. Calcd forC₄₈H₄₂N₄O₁₂F₆.3.1 H₂O: C, 55.61; H, 4.69; N, 5.40. Found: C, 55.24; H,4.36; N, 5.36.

Method Examples

COMPARE screening. The compounds described herein were examined forantiproliferative activity against the human cancer cell lines in theNational Cancer Institute screen (COMPARE screening), in which theactivity of each compound was evaluated with approximately 55 differentcancer cell lines of diverse tumor origins. The GI50 values (i.e., theconcentration causing 50% growth inhibition) obtained with selected celllines, along with the mean graph midpoint (MGM) values, are summarizedin Table 1 and Table 2, and provide a means of comparison of theantiproliferative activity of the compounds described herein with thatof other compounds, including camptothecin (S-1), oracin (S-2), and/or5,6-dihyro-6-(3-amino-1-propyl)-5,11-dioxo-11H-indeno[1,2,c]isoquinoline(S-3). The MGM is based on a calculation of the average GI50 for all ofthe cell lines tested (approximately 55) in which GI50 values below andabove the test range (10⁻⁸ to 10⁻⁴ molar) are taken as the minimum (10⁻⁸molar) and maximum (10⁻⁴ molar) drug concentrations used in thescreening test. Therefore, the MGM value represents an overallassessment of toxicity of the compound across numerous cell lines. Theresults of topoisomerase I DNA cleavage experiments are expressedsemiquantitatively and provide a means of comparison with the biologicalactivity of other compounds, including camptothecin (S-1) (++++), oracin(S-2) (+), and/or5,6-dihyro-6-(3-amino-1-propyl)-5,11-dioxo-11H-indeno[1,2,c]isoquinoline(S-3).

Hollow Fiber Activity. Several of the more active indenoisoquinolineanalogs (5p, 5q, 5s, 5v, and 5w) and several of the most activebisindenoisoquinoline analogs (14d, 14g, 14h, and 14i) were evaluated asanticancer agents in an in vivo animal model in which polyvinylidenefluoride (PVDF) “hollow fibers” containing various cancer cell cultureswere implanted intraperitoneally (IP) and subcutaneously (SC) intoathymic nude mice and compounds were administered by the IP route. Theeffects of the compounds on the reduction of viable cancer cell masscompared to those of controls were determined. Each compound was testedin the hollow fiber assay against a panel of twelve human tumor celllines as described previously; see, Hollingshead, M.; Plowman, J.;Alley, M.; Mayo, J.; Sausville, E., The Hollow Fiber Assay. Contrib.Oncol. 1999, 54, 109-120; and Plowman, J.; Carnalier, R.; Alley, M.;Sausville, E.; Schepartz, S. Contrib. Oncol. 1999, 54, 121-135, thedisclosures of which are hereby incorporated by reference. The compoundswere solubilized in 10% DMSO in saline/Tween-80® and administeredintraperitoneally once daily for a total of four doses at each of twodose levels. The two doses were selected based on single dose toxicitystudies for each derivative. A score of 2 was assigned each time thecompound produced a 50% or greater reduction in viable cell masscompared to vehicle-treated controls. The score for each compound wassummed for the intraperitoneal fibers and the subcutaneous fibers toprovide the total score for each derivative as shown in Table 3 andTable 4. For comparative purposes, the score for the clinically usedanticancer drug paclitaxel is provided.

Induction of DNA cleavage. The compounds described herein may beexamined for induction of DNA cleavage in the 3′-end-labeledPvuII/HindIII fragment of pBluescript SK(−) phagemid DNA in the presenceof top1 (see, Kohlhagen et al. “Protein-Linked DNA Strand Breaks Inducedby NSC 314622, a Novel Noncamptothecin Topoisomerase I Poison,” Mol.Pharmacol. 1998, 54, 50-58). The cleavage patterns for the compoundsdescribed herein can be determined, along with those of comparativecompounds NSC 314622 (A) (see, Kohlhagen et al., “Protein-Linked DNAStrand Breaks Induced by NSC 314622, a Novel NoncamptothecinTopoisomerase I Poison,” Mol. Pharmacol. 1998, 54, 50-58), camptothecin(B, CPT), and NSC 706744 (C, MJ-III-65) (see, Cushman et al., “Synthesisof New Indeno[1,2-e]isoquinolines: Cytotoxic Non-CamptothecinTopoisomerase I Inhibitors,” J. Med. Chem. 2000, 43, 3688-3698 andAntony et al., “Differential Induction of Topoisomerase I-DNA CleavageComplexes by the Indenoisoquinoline MJ-III-65 (NSC 706744) andCamptothecin: Base Sequence Analysis and Activity againstCamptothecin-Resistant Topoisomerase I,” Cancer Res. 2003, 63,7428-7435).

Topoisomerase 1-Mediated DNA Cleavage Reactions Using 3′-End-labeled 161BP Plasmid DNA. The 161 by fragment from pBluescript SK(−) phagemid DNA(Stratagene, La Jolla, Calif.) is cleaved with the restrictionendonuclease Pvu II and Hind III (New England Biolabs, Beverly, Mass.)in supplied NE buffer 2 (10 μL reactions) for 1 h at 37° C., separatedby electrophoresis in a 1% agarose gel made in 1×TBE buffer. The 161 byfragment is eluted from the gel slice (centrilutor by Amicon) andconcentrated in a centricon 50 centrifugal concentrator (Amicon,Beverly, Mass.). Approximately 200 ng of the fragment is 3′-end-labeledat the Hind III site by fill-in reaction with [alpha-³²P]-dCTP and 0.5mM dATP, dGTP, and dTTP, in React 2 buffer (50 mM Tris-HCl, pH 8.0, 100mM MgCl, 50 mM NaCl) with 0.5 units of DNA polymerase I (Klenowfragment). Labeling reactions are followed by phenol-chloroformextraction and ethanol precipitation. The resulting 161 by 3′-endlabeledDNA fragment is resuspended in water. Aliquots (approximately 50,000dpm/reaction) are incubated with topoisomerase I at 30° C. for 15 min inthe presence the compounds described herein. Reactions are terminated byadding 0.5% SDS. After ethanol precipitation, the samples areresuspended in loading buffer (80% formamide, 10 mM sodium hydroxide, 1mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue, pH 8.0),and separated in a denaturing gel (16% polyacrylamide, 7 M urea) run at51° C. The gel is dried and visualized by using a Phosphoimager andImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).

Topoisomerase II-Mediated DNA Cleavage Assays Using 5′-End-labeled HumanC-myc DNA. A 403-base pair DNA fragment of the human c-myc gene from thejunction between the first intron and the first exon is prepared by PCRbetween positions 2671 and 3073 using the a sense primer oligonucleotideand an antisense primer oligonucleotide, as described by Cushman et al.,in U.S. Pat. No. 6,509,344. Single-end labeling of these DNA fragmentsis obtained by 5′-end labeling of the adequate primer oligonucleotide.Approximately 0.1 μg of the human c-myc DNA that had been restricted byXhoI and XbaI is used as template for PCR. The 5′-end-labeled DNAfragments are equilibrated with or without a drug in 1% dimethylsulfoxide, 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl₂, 2 mMdithiothreitol, 0.1 mM Na₂EDTA, 1 mM ATP, and 15 μg/mL bovine serumalbumin for 5 min before addition of purified human topoisomerase II(40-70 ng) in a 10 μL final reaction volume. The reactions are performedat 37° C. for 30 min and thereafter stopped by adding 1% sodium dodecylsulfate (SDS) and 0.4 mg/mL proteinase K (final concentrations) followedby an additional incubation at 50° C. for 30 min. Samples areethanol-precipitated before separation of the topoisomerase II-cleavedfragments on denaturing polyacrylamide gels. The sequencing gels aremade of 7% polyacrylamide in IX TBE buffer (90 mM Tris borate, 2 mMEDTA, pH 8.3). Electrophoresis is performed at 2500 V (60 W) for 2-5 h.The gels were dried and visualized using a Phosphoiniager and ImageQuantsoftware.

DNA Cleavage Semiquantitative Analysis. One of the most abundantcleavage products (see, Antony et al., “Differential Induction ofTopoisomerase I-DNA Cleavage Complexes by the IndenoisoquinolineMJ-III-65 (NSC 706744) and Camptothecin: Base Sequence Analysis andActivity against Camptothecin-Resistant Topoisomerase I,” Cancer Res.2003, 63, 7428-7435) is chosen for semiquantitation using ImageQuant TLv2003.3. The rubberband baseline correction is applied with banddetection sensitivity set at 90. In the case of the compounds describedherein, the absolute density value for the band corresponding to theabove product is compared to the value for the NSC 314622 (A). The ratioof the band density observed for the compounds described herein to theNSC 314622 band is multiplied by 100 to obtain percentages. Assignmentsare performed as follows: 0-25%, 0; 25-75%, +; 75-175%, ++; 175-325%,+++; camptothecin ++++.

SV40 DNA Unwinding Assay. Reaction mixtures (10 μL final volume) contain0.3 μg supercoiled SV40 DNA in reaction buffer (10 mM Tris-HCl, pH 7.5,50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 15 μg/mL bovine serum albumin) and10 units of purified calf thymus topoisomerase I. Reactions areperformed at 37° C. for 30 min and terminated by the addition of 0.5%SDS, and then 1.1 μL of 10× loading buffer (20% Ficol 400, 0.1 M Na₂EDTApH 8, 1.0% SDS, 0.25% Bromophenol Blue) is then added and reactionmixtures are loaded onto a 1% agarose gel made in IX TBE buffer. Afterelectrophoresis, DNA bands are stained in 10 μg/mL of ethidium bromideand visualized by transillumination with UV light (300 nm).

Additional details regarding the biological evaluation of the compoundsdescribed herein may be found in co-pending PCT/US2005/008491, thedisclosure of which is incorporated herein by reference.

TABLE 1 Cytotoxicities and Topoisomerase I Inhibitory Activities ofIndenoisoquinoline Analogs. cytotoxicity (GI50 in μM)^(a) colon lungHCT- CNS melanoma ovarian renal prostate breast Top 1 Cmpd HOP-62 116SF-539 UACC-62 OVCAR-3 SN12C DU-145 MDA-MB-435 MGM^(b) Cleavage^(c) S-10.01 0.03 0.01 0.01 0.22 0.02 0.01 0.04  .0405 ± 0.0187 ++++ S-2 1.621.12 1.65 1.42 3.85 0.95 1.28 2.56 1.90 ± 0.80 + 4a NT 100 100 100 100100 100 100 100 ++ 4b 53.7 >100 >100 >100 >100 >100 >100 >100 57.5 ++ 4c18.20 47.9 >100 25.1 >100 >100 >100 >100 64.6 0/+4d >100 >100 >100 >100 >100 >100 >100 >100 >100 0 5a NT 2.45 6.17 6.615.89 11.0 4.47 7.08 6.17 ++ 5b <0.010 <0.010 2.69 0.30 2.63 0.023 2.043.02 0.525 0/+ 5c 5.62 6.46 NT 7.08 25.7 4.17 5.62 >100 9.77 +++ 5d 1.740.58 1.86 0.51 1.70 0.91 1.32 2.82 1.86 +++ 5h 89.1 60.3 >10056.2 >100 >100 >100 >100 741 +++ 5i 52.50 >100 NT 83.2 >100 58.961.7 >100 74.1 ++++ 5j >100 36.3 85.1 29.5 81.3 93.3 >100 >100 67.6 ++++5e <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 0.014 0.033 +++ 5f0.19 0.274 0.016 0.012 0.864 0.015 0.017 2.17 0.370 ± 0.28  ++++ 5g 2.691.41 2.34 0.79 1.66 1.66 1.41 2.75 1.86 +++++ 5l <0.010 <0.010 0.037<0.010 0.085 <0.010 <0.010 0.020 0.079 ± 0.023 ++++ 5m 0.447 1.99 0.3980.269 56.2 0.316 0.363 7.08 2.16 ± 0.24 ++ 5n 0.079 1.91 0.288 <0.01061.7 0.085 0.085 >100 3.55 +++ 5o <0.010 <0.010 <0.010 0.014 0.041<0.010 <0.010 <0.010 0.112 ± 0.066 ++++ 5p <0.005 0.575 <0.005 1.20 2.040.091 0.015 4.57 0.382 ± 0.119 ++ 5q 1.78 1.15 0.040 0.030 74.1 0.8130.155 67.6 4.64 ± 1.25 ++++ 5r 26.3 72.4 18.2 37.2 34.7 NT >100 >10050.1 ++ 5s <0.005 <0.005 <0.005 5.01 5.75 0.126 <0.005 0.977 0.243 ±0.088 +++ 5t 18.2 1.48 17.8 15.1 15.1 11.5 10.7 >100 12.0 + 5u 0.4270.120 0.100 1.29 0.832 0.257 0.182 1.74 0.766 ± 0.254 + 5v <0.005 0.2140.145 0.457 5.01 0.145 0.081 2.63 0.715 ± 0.335 +++ 5w 9.77 2.34 1.441.23 15.1 >100 0.275 >100 7.86 ± 0.27 ++ 5x <0.005 <0.005 0.550 0.1620.525 1.48 0.603 1.95 1.27 ± 0.84 +++ 5z 24.5 >50.128.2 >50.1 >50.1 >50.1 >50.1 >50.1 39.8 0 5af NT 17.4 20.9 20.0 33.974.1 31.6 81.3 30.9 0 5ag 10.7 25.7 8.71 15.5 — >50.1 >50.1 >50.1 19.9 05ah 24.5 NT 17.8 17.4 28.8 77.6 >100 >100 50.1 − 5ai 28.8 60.3 42.743.6 >100 >100 97.7 >100 52.5 0 5aj 20.9 35.5 29.5 24.0 70.8 >10091.2 >100 42.6 ± 5.35 − 5ak 32.4 >100 20.4 27.5 >100 >100 91.2 >100 51.3− 5al 0.620 0.270 0.210 0.920 0.710 0.490 0.760 0.920 0.530 ± 0.320 +++5am 0.200 0.180 0.25 0.26 1.38 0.160 0.22 0.78 0.32 ± 0.23 +++ 5an 0.080.10 0.10 0.05 0.52 0.04 0.01 0.84 0.16 ± 0.01 +++ 5ao 0.288 0.200 0.8711.35 0.708 0.398 0.347 1.35 0.471 ± 0.054 0 5ap 1.29 0.912 1.23 1.622.00 1.32 0.603 2.04 1.32 ++ 5aq 1.20 1.26 1.78 2.00 1.70 1.66 0.8322.24  1.66 ± 0.155 0 5ar 2.14 1.66 2.82 3.80 3.47 3.47 3.39 5.62 3.71 +5as 6.76 6.46 9.77 9.33 9.55 8.51 6.03 9.55 8.13 0 5at 4.79 2.75 1.8213.8 10.7 3.31 3.47 11.7 5.50 0 5au 2.19 1.91 2.04 1.70 2.09 2.00 4.5711.0 5.13 + 5av 17.0 NT 18.2 14.8 17.8 13.5 19.1 19.5 18.2 0/+ 5aw 11.2NT 12.3 10.7 13.8 28.2 13.5 3.09 15.1 0 5ax 7.59 NT 6.76 8.71 13.8 13.813.8 42.7 11.5 +++ 5ay 21.4 NT 17.4 27.5 >100 77.6 74.1 5.13 53.7 0 5az28.8 NT 27.5 89.1 >100 >100 81.3 4.57 44.7 0 5ba 0.295 0.794 0.027<0.010 3.39 <0.010 0.036 3.24 0.178 ± 0.012 ++++ 5bb NT3.47 >100 >100 >100 >100 >100 >100 40.0 0 5bc NT NT NT NT NT NT NT NT NT+++ 5bd NT 0.046 0.058 0.148 3.02 0.309 0.034 1.48 0.328 ± 0.046 ++++5bf 33.9 26.9 44.7 75.9 52.5 >100 61.7 64.6 38.9 +++ 5bg <0.010 <0.0100.038 NT 0.028 <0.010 0.014 0.059 0.048 ± 0.024 + 5bh 7.59 4.90 NT 19.57.94 25.1 29.5 7.76 12.3 0 5bi 0.021 0.038 0.095 0.380 NT 0.309 0.0851.23 0.632 ± 0.029 +++ 5bj <0.010 <0.010 NT <0.010 <0.010 <0.010 NT<0.010 0.014 ± 0.001 NA 5bk 1.41 1.26 1.95 1.58 2.69 4.07 2.29 4.68 2.70 ± 0.125 + 5bl 0.031 0.027 >100 0.200 1.35 0.229 >100 1.07 0.296 ±0.067 NA 5bm <0.010 NT <0.010 <0.010 <0.010 0.012 <0.010 <0.010 0.016++++ 5bn 0.026 0.044 0.112 0.550 0.417 0.158 0.055 0.389 0.124 ± 0.014 05bo 0.195 NT 0.550 0.178 0.550 0.269 0.174 0.490 0.339 NA 5bp <0.010<0.010 <0.010 <0.010 0.028 <0.010 <0.010 <0.010 0.020 ± 0.001 NA 5bq0.078 0.102 0.240 1.00 0.427 0.245 0.257 0.617 0.300 ± 0.072 0 5br<0.010 <0.010 <0.010 <0.010 0.020 <0.010 <0.010 <0.010 0.019 ± 0.004 NA5bs 0.056 0.110 0.178 0.071 1.66 0.676 0.204 0.646 0.416 ± 0.134 +++^(a)The cytotoxicity GI50 values are the concentrations corresponding to50% growth inhibition. ^(b)Mean graph midpoint for growth inhibition ofall human cancer cell lines successfully tested. ^(c)The compounds weretested at concentrations ranging up to 10 μM. The activity of thecompounds to produce top 1-mediated DNA cleavage was expressedsemiquantitatively as follows: +: weak activity; ++ and +++: modestactivity; ++++: similar activity as 1 μM camptothecin; +++++: greateractivity than 1 μM camptothecin. NT: Not Tested; NA: Not Available S-1 =camptothecin S-2 = oracin

TABLE 2 Cytotoxicities and Topoisomerase I Inhibitory Activities ofBis-Indenoisoquinoline Analogs. cytotoxicity (GI50 in μM)^(a) lung colonCNS melanoma ovarian renal prostate breast Top 1 Cmpd HOP-62 HCT-116SF-539 UACC-62 OVCAR-3 SN12C DU-145 MDA-MB-435 MGM^(b) Cleavage^(c) S-30.20 0.18 0.25 0.26 1.38 0.16 0.22 0.78 0.32 ± 0.23 +++ S-1 0.01 0.030.01 0.01 0.22 0.02 0.01 0.04  .0405 ± 0.0187 ++++12a >25.1 >25.1 >25.1 >25.1 >25.1 >25.1 >25.1 >25.1 18.2 + 12c 0.7940.550 3.63 6.61 2.95 1.55 1.00 8.91 4.28 ± 1.89 + 12d NT NT 1.12 2.001.20 0.589 NT 1.55 0.934 ± 0.476 ++ 13a 22.4 22.9 >50.1 >50.121.4 >50.1 >50.1 >50.1 33.9 0 13b 20.0 14.1 >50.1 45.7 13.239.8 >50.1 >50.1 28.2 0 13d 11.0 1.91 8.13 93.3 69.2 36.3 47.9 69.2 35.5++ 14a 0.977 1.05 14.5 5.01 8.91 11.0 1.91 2.24 5.25 + 14b 0.028 0.056NT 0.513 0.372 0.132 0.288 0.562 0.357 ± 0.087 + 14c 0.032 0.029 NT0.331 1.66 0.178 0.182 1.66 0.427 ± 0.01  + 14d 0.339 <0.005 0.155 0.1820.093 <0.005 0.079 0.024 0.122 ± 0.064 ++++ 14e <0.010 <0.010 NT 0.0521.02 <0.010 <0.010 0.933 0.152 ± 0.062 ++++ 14f 12.9 35.5 >100 >100 >10015.5 24.0 >100 44.8 ± 2.05 0 14g <0.010 <0.010 0.011 0.042 0.074 <0.010NT 0.107 0.394 ± 0.33  ++++ 14h 0.525 <0.005 0.251 0.562 0.135 <0.0050.234 0.676 0.225 ± 0.084 +++ 14i 0.048 0.112 0.275 0.269 1.15 0.0170.331 1.00 0.474 ± 0.143 +++ 14j 0.977 0.200 0.012 NT 0.032 NT 0.0850.126 0.262 ± 0.100 ++ 14k 0.068 0.045 0.170 1.23 0.269 0.028 0.2090.813 0.562 ++ 14l 1.51 0.331 4.17 4.27 9.55 0.240 19.5 3.98 6.03 ++ 14m0.631 0.044 0.324 0.603 0.245 0.123 0.813 0.437 0.354 ± 0.184 ++ 14n3.02 1.45 1.17 1.78 2.29 1.17 0.912 3.89 1.50 ± 0.24 0 A^(d) NT 43 >10044 0.88 33 >100 68 58.9 ++ 16a >100 >100 NT >100 NT >100 NT >100 68.0 017a 0.191 0.022 <0.010 NT <0.010 NT <0.010 0.155 0.046 ± 0.010 + ^(a)Thecytotoxicity GI50 values are the concentrations corresponding to 50%growth inhibition. ^(b)Mean graph midpoint for growth inhibition of allhuman cancer cell lines successfully tested. ^(c)The compounds weretested at concentrations ranging up to 10 μM. The activity of thecompounds to produce top1-mediated DNA cleavage was expressedsemi-quantitatively as follows: + & ++: weak activity; +++: similaractivity as compound S-3; ++++: similar activity as 1 μM camptothecin;NT: Not Tested. S-1 = camptothecin; S-3 =5,6-dihyro-6-(3-amino-1-propyl)-5,11-dioxo-11H-indeno[1,2,c]isoquinoline (NSC 725671). ^(d)A =5,6-dihyro-2,3-dimethoxy-8,9-methylenedioxy-6-(3-aminopropylaminopropylamino-1-propyl)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline

TABLE 3 Hollow Fiber Activities of Indenoisoquinoline Analogs. CompoundIP Score^(a) SC score^(a) Total score Cell kill^(b) 5p 16 2 18 N 5q 4 26 N 5s 6 0 6 N 5v 12 2 14 N 5w 8 0 8 N Paclitaxel 24 8 32 Y ^(a)The IPand SC scores listed are the sums of all the IP and SC scores for eachcompound. ^(b)A net cell kill at one or more implant sites is indicatedwith a Y.

TABLE 4 Hollow Fiber Activities of Bis-Indenoisoquinoline Analogs.Compound IP Score^(a) SC score^(a) Total score Cell kill^(b) 14d 2 4 6 N14g 26 6 32 N 14h 12 4 16 N 14i 10 6 16 N Paclitaxel 24 8 32 Y ^(a)TheIP and SC scores listed are the sums of all the IP and SC scores foreach compound. ^(b)A net cell kill at one or more implant sites isindicated with a Y.

What is claimed is:
 1. A pharmaceutical composition for treating cancer,the composition comprising: (a) a compound of the formula

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein: m is an integer from 0 to about 6; R⁶ is selected from thegroup consisting of haloalkyl, halocycloalkyl, hydroxy, alkoxy,cycloalkoxy, haloalkoxy, halocycloalkoxy, optionally substitutedheteroaryl, aryloxy, heteroaryloxy, and heteroarylamino, acyloxy,haloacyloxy, amino, alkyl and dialkylamino, trialkylammonium,bis(hydroxyalkyl)amino, hydroxyalkylaminoalkylamino,heteroarylalkylaminoalkylamino, acylamino, hydroxylamino, alkoxylamino,acyloxylamino, cycloalkyl, heterocyclyl, heterocyclylamino, alkynyl,acyl, urethanyl, cyano, nitro, azido, thio, alkylsulfonyl, sulfonic acidand derivatives thereof, carboxylic acid and derivatives thereof, andphosphonic acid and derivatives thereof; provided that when R⁶ ishydroxy, alkylamino, or hydroxyalkylamino, m is the integer 0; R^(a)represents 1-4 substituents each of which is independently selected fromthe group consisting of hydrogen, halo, hydroxy, optionally substitutedalkyl, optionally substituted alkoxy, cyano, nitro, optionallysubstituted alkylthio, optionally substituted alkylsulfonyl, carboxylicacid and derivatives thereof, and sulfonic acid and derivatives thereof;or R^(a) represents 2-4 substituents where 2 of said substituents areadjacent substituents and are taken together with the attached carbonsto form an optionally substituted heterocycle, and where any remainingsubstituents are each independently selected from the group consistingof hydrogen, halo, hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy, cyano, nitro, optionally substituted alkylthio,optionally substituted alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof; R^(d) represents 1-4substituents each of which is independently selected from the groupconsisting of hydrogen, halo, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, cyano, nitro, optionally substitutedalkylthio, optionally substituted alkylsulfonyl, carboxylic acid andderivatives thereof, and sulfonic acid and derivatives thereof; or R^(d)represents 2-4 substituents where 2 of said substituents are adjacentsubstituents and are taken together with the attached carbons to form anoptionally substituted heterocycle, and where any remaining substituentsare each independently selected from the group consisting of hydrogen,halo, hydroxy, optionally substituted alkyl, optionally substitutedalkoxy, cyano, nitro, optionally substituted alkylthio, optionallysubstituted alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof; and (b) one or morepharmaceutically acceptable carriers, diluents, and excipients therefor;where the compound is present in an amount effective for treating acancer in a patient in need of relief. 2-10. (canceled)
 11. A compoundof the formula

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein: m is an integer from 0 to about 6; R⁶ is selected from thegroup consisting of haloalkyl, halocycloalkyl, hydroxy, alkoxy,cycloalkoxy, haloalkoxy, halocycloalkoxy, optionally substitutedheteroaryl, aryloxy, heteroaryloxy, and heteroarylamino, acyloxy,haloacyloxy, amino, alkyl and dialkylamino, trialkylammonium,bis(hydroxyalkyl)amino, hydroxyalkylaminoalkylamino,heteroarylalkylaminoalkylamino, acylamino, hydroxylamino, alkoxylamino,acyloxylamino, cycloalkyl, heterocyclyl, heterocyclylamino, alkynyl,acyl, urethanyl, cyano, nitro, azido, thio, alkylsulfonyl, sulfonic acidand derivatives thereof, carboxylic acid and derivatives thereof, andphosphonic acid and derivatives thereof; provided that when R⁶ ishydroxy, alkylamino, or hydroxyalkylamino, m is the integer 0; R^(a)represents 1-4 substituents each of which is independently selected fromthe group consisting of hydrogen, halo, hydroxy, optionally substitutedalkyl, optionally substituted alkoxy, cyano, nitro, optionallysubstituted alkylthio, optionally substituted alkylsulfonyl, carboxylicacid and derivatives thereof, and sulfonic acid and derivatives thereof;or R^(a) represents 2-4 substituents where 2 of said substituents areadjacent substituents and are taken together with the attached carbonsto form an optionally substituted heterocycle, and where any remainingsubstituents are each independently selected from the group consistingof hydrogen, halo, hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy, cyano, nitro, optionally substituted alkylthio,optionally substituted alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof; and R^(d) represents1-4 substituents each of which is independently selected from the groupconsisting of hydrogen, halo, hydroxy, optionally substituted alkyl,optionally substituted alkoxy, cyano, nitro, optionally substitutedalkylthio, optionally substituted alkylsulfonyl, carboxylic acid andderivatives thereof, and sulfonic acid and derivatives thereof; or R^(d)represents 2-4 substituents where 2 of said substituents are adjacentsubstituents and are taken together with the attached carbons to form anoptionally substituted heterocycle, and where any remaining substituentsare each independently selected from the group consisting of hydrogen,halo, hydroxy, optionally substituted alkyl, optionally substitutedalkoxy, cyano, nitro, optionally substituted alkylthio, optionallysubstituted alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof. 12-31. (canceled)
 32. A compoundof the formula

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein: X is a divalent linker comprising one or more divalent radicalsselected from the group consisting of —(CR¹R²)—, —(NR¹)— and —O—, whereR¹ and R² are independently selected in each occurrence from hydrogen,alkyl, and acyl, providing that the divalent linker does not include thedivalent radical —O—O—; R^(a) and R^(a′) each represent 1-4 substituentseach of which is independently selected from the group consisting ofhydrogen, halo, hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy, cyano, nitro, optionally substituted alkylthio,optionally substituted alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof; or R^(a) and R^(a′)each represent 2-4 substituents where 2 of said substituents areadjacent substituents and are taken together with the attached carbonsto form an optionally substituted heterocycle, and where any remainingsubstituents are each independently selected from the group consistingof hydrogen, halo, hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy, cyano, nitro, optionally substituted alkylthio,optionally substituted alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof; and R^(d) and R^(d)′each represent 1-4 substituents each of which is independently selectedfrom the group consisting of hydrogen, halo, hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy, cyano, nitro,optionally substituted alkylthio, optionally substituted alkylsulfonyl,carboxylic acid and derivatives thereof, and sulfonic acid andderivatives thereof; or R^(d) and R^(d′) each represents 2-4substituents where 2 of said substituents are adjacent substituents andare taken together with the attached carbons to form an optionallysubstituted heterocycle, and where any remaining substituents are eachindependently selected from the group consisting of hydrogen, halo,hydroxy, optionally substituted alkyl, optionally substituted alkoxy,cyano, nitro, optionally substituted alkylthio, optionally substitutedalkylsulfonyl, carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof. 33-42. (canceled)
 43. A method fortreating cancer, the method comprising the step of administering atherapeutically effective amount of a compound of claim 11 to a patientin need of relief from said cancer.
 44. A process for preparing acompound of the formula

without isolating an intermediate product, the process comprising:reacting a compound of the formula

with a compound of the formula

and cyclizing the intermediate product wherein R^(a) and R^(d) are asdefined in claim
 1. 45. The process of claim 44 wherein the cyclizingstep includes one or more acids.
 46. The process of claim 44 wherein thecyclizing step includes dicyclohexylcarbodiimide.
 47. A process forpreparing a compound of claim 11, the process comprising the step ofreacting a compound of the formula

with an amine of the formula R⁶—(CH₂)_(m)—NH₂, wherein R^(a), R^(d), m,and R⁶ are as defined in claim
 11. 48. A process for preparing acompound of claim 32, the process comprising the step of reacting acompound of the formula

with a polyamine of the formulaNH₂—(CH₂)_(n)—[(CH₂)_(x)—NR¹—(CH₂)_(y)]_(z)—(NR²)_(p)—(CH₂)_(q)—NH₂,wherein R¹, R², n, x, y, z, p, q, R^(a), and R^(d) are as defined inclaim
 33. 49-51. (canceled)
 52. The process of claim 48 whereinR^(a)═R^(a′) and R^(d)═R^(d′), and said process is performed in onestep.